|Year : 2018 | Volume
| Issue : 1 | Page : 27-32
TNF-α-mediated inflammation and cytotoxicity of different root canal sealers and root-end fillings in cell line culture
Mohamed I Elshinawy1, Hossam A Eid2, Ashraf A Khalil3, Abdelnasser M Soliman4, Hesham El-Refaey5
1 Department of Endodontics, Faculty of Dentistry, Tanta University, Tanta, Egypt
2 Department of Oral Medicine and Periodontology, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt; Department of Periodontics, College of Dentistry, Gulf Medical University, Ajman, UAE
3 Department of Crown and Bridge, College of Dentistry, Tanta University, Tanta, Egypt
4 Department of Oral Biology, College of Oral and Dental Medicine, Cairo University, Egypt; Department of Oral and Maxillofacial Surgery and Diagnostic Sciences, College of Dentistry, King Khalid University, Abha, KSA
5 Department of Pharmacology, College of Pharmacy, Al Azhar University, Cairo, Egypt
|Date of Submission||20-Sep-2017|
|Date of Acceptance||28-Dec-2017|
|Date of Web Publication||4-Apr-2018|
Mohamed I Elshinawy
Department of Restorative Dental Sciences, College of Dentistry, King Khalid University, PO Box 3263, Abha 61471, Saudi Arabia
Source of Support: None, Conflict of Interest: None
The aim was to detect the amount of tumor necrosis factor-α (TNF-α) released from Hela epithelial cells as a measure of the inflammatory and cytotoxicity impact of different root end filling and sealant materials on the apical periodontal tissues.
Materials and methods
Standardized discs of Amalgam (G1), composite (G2), flowable composite (G3), zinc oxide eugenol (ZOE) sealer (G4), nano-ZOE sealer (G5), and resin-modified glass ionomer (G6) were incubated with Hela cells. The inflammatory and biological cytotoxicity of the nominated materials were evaluated in terms of the amount of TNF-α released from Hela cells and its effect on cellular morphology and viability in comparison to non-treated cells (control).
Both composite root-end filling and nano-ZOE sealer showed non-significant difference in the amount of released TNF-α compared to that of the control (P > 0.05). Accordingly, these materials showed non-significant effect on Hela-cell viability and morphology. On the other hand the remaining materials demonstrated significant increase in the amount secreted TNF-α associated and hence significant cytotoxic effects in comparison to that of the control (P > 0.05)
Composite would be the recommended filling for root-end cavities and nano-ZOE is a promising biocompatible sealer material.
Keywords: hela epithelial cells, root canal sealers, root end filling materials, tumor necrosis factor-α
|How to cite this article:|
Elshinawy MI, Eid HA, Khalil AA, Soliman AM, El-Refaey H. TNF-α-mediated inflammation and cytotoxicity of different root canal sealers and root-end fillings in cell line culture. Tanta Dent J 2018;15:27-32
|How to cite this URL:|
Elshinawy MI, Eid HA, Khalil AA, Soliman AM, El-Refaey H. TNF-α-mediated inflammation and cytotoxicity of different root canal sealers and root-end fillings in cell line culture. Tanta Dent J [serial online] 2018 [cited 2018 Dec 12];15:27-32. Available from: http://www.tmj.eg.net/text.asp?2018/15/1/27/229246
| Introduction|| |
Biocompatibility of materials used in dentistry is of prime importance. Leached out components of some of restorative materials have shown some unfavorable tissue response and inflammatory reactions ,,,,.
Root-end fillings and root canal sealants should be stable dimensionally, moisture and water insoluble, adhesive to dentinal walls and biologically inert. Among the materials used for these purposes are amalgam, composite, glass ionomer, zinc oxide eugenol (ZOE) and more recently nano-ZOE sealer has been introduced for its claimed antimicrobial activity ,,,,.
Cytokines are proteins of soluble nature capable of mediating cellular response through special cell membrane receptors. Their effect is either proinflammatory or anti-inflammatory depending on the surrounding environment that might have either cooperating or competing factors that affect the cytokine receptors and signal interaction with the target cells . Tumor necrosis factor-α (TNFα) is a strong immune-modulator and proinflammatory cytokine produced mainly by macrophages or monocytes and to less extent by keratinocytes, lymphocytes, and fibroblasts ,,,.
Although the cytotoxicity of various dental materials was under the focus of investigations for many years, still few is available regarding the potential effect of recently introduced nanodental materials on cell viability, inflammation, necrosis, immortalization, and apoptosis compared to the traditional ones. Some material components of the retrograde fillings or root canal sealers might leach into the periapical tissues during various conventional and surgical endodontic procedures with a potential harmful effect . A detailed signaling pathway triggered by these dental materials should be elucidated to explore their potential activities in the physiological circumstances of living subjects. To address these points, we attended to measure the proinflammatory mediator TNF-α as an inflammation indicator. The null hypothesis of this study was that all the tested materials are of comparable cytotoxicity and capability to provoke TNF-α-mediated inflammation.
| Materials and Methods|| |
Preparation of standardized dental restorative materials discs
This study was conducted following the ethical approval of the Scientific and Research Committee, College of Dentistry, King Khalid University. A brass mold (6 mm diameter and 2 mm height) was used for preparation of standardized discs from the dental materials tested in the study before its incubation with Hela cells (Hela-Cell Line human; Sigma, St Louis, Missouri, USA). The materials used were as following: composite (Filtek Z250, 3M, ESPE; Dentsply Products, St Paul, Missouri, USA), resin-modified glass ionomer (RMGI) (3M ESPE, Bangalore, India, Photac Fil Quick Aplicap, 3M; St Paul, Minnesota, USA), flowable composite (Tetric N-Flow; Ivoclar Vivadent GmbH, Donau, Austria), ZOE sealer (Endofil; Promedica, Neumünster, Germany), amalgam (Megalloy EZ; Dentsply Detrey GmbH, Konstanz, Germany), and nano-ZOE sealer (ZO nanopowder; Sigma-Aldrich). All of the above mentioned materials were manipulated according to their manufacturer's instructions.
Hela cells were cultured in minimal essential medium (MEM/H; Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Invitrogen, Carlsbad, California, USA), 2 mM l-glutamine (Gibco; Invitrogen), and 1 μg/ml of Gibco antibiotic-antimycotic (Gibco; Invitrogen) to prevent bacterial and fungal contamination. This solution contains 10 000 U/ml of penicillin, 10 000 μg/ml of streptomycin, and 25 μg/ml of Gibco Amphotericin B.
The antibiotics penicillin and streptomycin prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria. Amphotericin B prevents fungal contamination of cell cultures due to its inhibition of multicellular fungus and yeast. The cells were maintained at 37°C in a humidified incubator containing 5% CO2 for 48 h.
Discs of the six nominated materials were incubated for 48 h with Hela cells culture in well plates at 37 ± 1°C. Another plate having only Hela cells culture was also incubated for control purposes.
The cells were thereafter photographed at ×40 under an inverted light microscope (Nikon, Eclipse E100; Nikon Corporation, Konan, Minato-Ku, Tokyo, Japan) to determine the changes in general morphological structure of cells in response to the tested materials.
Determination of cell viability by trypan blue assay
Trypan Blue (Sigma-Aldrich) is a vital dye and its reactivity is due to the negatively charged chromopore that interact only with cells having damaged membranes. This explains why only the non-viable cells are stained thus the viable ones could be counted.
After 48 h of material-cell incubation was carried out at 37°C, cells were exposed to 0.2% trypan blue and cell count done in a haemocytometer (Bright-Line Hemacytometer; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The cell viability was assessed by trypan blue dye exclusion test . The adherent cells to the culture plates were washed with PBS and trypsinized (Trypsin-EDTA Solution; Sigma-Aldrich) to dissociate the cell clusters from the culture plates then re-suspended in a fresh minimal essential medium (Sigma). The cell suspension was mixed by pipetting several times to get uniform single cell suspension, then 100 μl of 0.4% trypan blue dye was added to 100 μl of the cell suspension. The content was mixed well and incubated for 5 min at room temperature to allow dead cells to take up the dye and the viable cells to pump out the stain by efflux mechanism. The cover slip was placed and 10 μl of the prepared trypan blue-cell suspension was transferred to haemocytometer.
The viable and non-viable cells were counted separately in all four squares using the formula: cell viability (%)=total viable cells (unstained)/total cells (stained and unstained)×100.
Determination of amount of released tumor necrosis factor-α
Hela cells cultured in a seven-well microplate (1 × 105 cells per well), six of them were co-incubated with the prepared test material discs for 48 h. at 37°C in 5% CO2 atmosphere and the seventh served as a control. The cells were then harvested by trypsinization and an equal aliquot of the cells of each group were used for the determination of TNF-α using enzyme-linked immunosorbent assay (ELISA) kit (Sigma-Aldrich Chemie GmbH). A specific monoclonal antibody for TNF-α has been coated onto the wells of the microtiter strips provided. Samples, including standards of TNF-α content, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the antigen bound to the immobilized antibody on one site. After washing, a biotinylated antibody specific was added. During the second incubation, this antibody bound to the immobilized antigen captured during the first incubation. After removal of excess second antibody, streptavidin-peroxidase was added. This bound to the biotinylated antibody to complete the four-member sandwich. After third incubation and washing to remove the entire unbound enzyme, a substrate solution 'which was acted upon by the bound enzyme to produce color' was added and incubated for 15–20 min followed by adding a stop solution for color development. Since the intensity of the developed color is in direct proportion to the concentration of the TNF-α produced in the Hela cells, the optical density of the developed color was measured for both control and test groups using an EnVision plate reader (PerkinElmer Inc., Waltham, Massachusetts, USA) at 450 nm with a reference filter of 570 nm . The amount of the TNF-α was then calculated in ng/ml from the standard curve .
Statistical analysis of the collected data was performed using version 19 Prism data analysis program (GraphPad Software Inc., San Diego, California, USA). Both one-way analysis of variance and Mann–Whitney tests were used to detect the differences among the test groups.
| Results|| |
After 48 h incubation, obvious adhesion and morphologic change of the Hela epithelial cells was noticed with amalgam, ZOE sealer, flowable composite and RMGI (groups 1, 3, 4, and 6). Normal cells changed to be spindle in shape with large oval nuclei. The proliferation rate of Hela cells was also reduced and the density of the cells was diminished. On the other hand, the other groups (groups 2 and 5) showed less morphological changes in comparison to the control [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f, [Figure 1]g.
|Figure 1: Morphological changes of Hela cells after 48 h exposure to the test materials. RMGI, resin-modified glass ionomer; ZOE, zinc oxide eugenol.|
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The inhibitory rate of cell viability was significantly increased in groups 1, 3, 4, and 6 in comparison to the control (P < 0.05). No difference in the inhibitory rate was determined between groups 2, 5, and the control (P > 0.05) [Table 1] and [Figure 2].
|Table 1: Mean values of the percentage of viable cells in response to test materials|
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|Figure 2: Effect of the test materials on Hela-cell viability. RMGI, resin-modified glass ionomer; ZOE, zinc oxide eugenol.|
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The mean and SD for the amount of released TNF-α in response to the tested materials are shown in [Table 2]. One-way analysis of variance of the results showed a significant difference among the tested groups at P < 0.05 (P = 9.609E − 12). The amount of the TNF-α secreted in groups 1, 3, 4, and 6 was higher than that recorded for the control (P < 0.05). The least amount of released TNF-α was recorded for both nano-ZOE Sealer (group 5) and dental composite (group 2) which was comparable to the control group (P > 0.05).
|Table 2: Mean values of the level of secreted tumor necrosis factor-α in Hela cells with different test materials|
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| Discussion|| |
Root-end fillings are materials used for sealing retro-cavities in apicectomized roots and remain in direct contact with periapical tissue. Root canal sealers are also essential part of root canal obturation and might be extruded periapically during endodontic treatment. These materials should have excellent sealing ability and biocompatibility to promote periapical healing ,.
Cytotoxicity was usually used as a measure of biocompatibility of different dental materials. However, recent research studies are focusing on their capability to support a favorable cellular response . In this study TNF-α release from Hela cells was used as a measure of the inflammatory response induced by some of the commonly used RCS and root end filling materials beside the newly introduced nano-ZOE sealer that might be widely used in the future because of its claimed therapeutic effect ,.
The results of the present study revealed that both dental composite and nano-ZOE were comparable to the control and exhibited less TNF-α releasing capabilities and hence less amount of cytotoxicity and less effect on cell viability compared to flowable composite, amalgam, ZOE sealer, and RMGI.
The excellent biocompatibility of nano-ZOE sealer in the current study is in agreement with Sousa et al.  study's conclusion that, nano-ZOE is well-tolerated, biocompatible allowing bone formation and remodeling. The favorable biological reaction of dental composite in our study is also supported by a previous study for assessment of the cytotoxicity of eight dental composite materials on cultured human fibroblasts and the authors concluded that all the tested composite materials were not cytotoxic to the living cells .
It has been shown that, out of more than 30 different compounds extracted from polymerized dental composites, its cytotoxicity was mainly attributed to the residual monomers release as a result of material degradation or incomplete polymerization ,,. It has also been shown that the removal of leachable components from polymerized composites by organic solvents completely decreased cytotoxicity . In addition, non-cured resinous materials were found more cytotoxic than cured and the effect varied according to aging of test specimens which reflected the role of free monomer in this regard . Previous supporting studies also revealed that, the high amount of resin in flowable composite is responsible for more production of TNF-α and hence its cytotoxicity ,,. The results of the above mentioned studies might explain the unfavorable biological response encountered with both flowable composite and RMGI in our study. In agreement, the cytotoxicity of RMGI was referred by another previous study both of its free monomer component and the released monomer during the mechanism of monomer-polymer conversion ,. Furthermore, RMGI was more cytotoxic than conventional glass ionomer and composite resin emphasizing the role of each particular component .
The unfavorable response encountered with ZOE sealer and amalgam in our study was supported by the findings of Huang et al.  who reported marked cytotoxicity of ZOE sealer on permanent V79 cells and human periodontal ligament cells. This cytotoxicity was found in another study to be more than that of resin sealer which produced its cytotoxicity through a genotoxic effect by breaking and digestion of genomic DNA . Moreover, some other investigations have proved a high amalgam cytotoxicity and referred this to the release of trace amounts of unreacted silver and mercury ,. However, another study showed a low cytotoxicity of amalgam on L929 cell line which was referred to the very little amount of leached trace elements . These results might also raise the importance of the type of the amalgam used and its composition together with the cell line used which might have a different response from that of Hela-cell line used in our study.
The results of TNF-α release levels in this study in response to different test materials were supported by the concomitant variable levels of cytotoxic effect of each of them on Hela-cell morphology viability results of this study where significantly less cell viability and more morphological changes were observed after 48 h of incubation with flowable composite, amalgam, and RMGI discs compared to nano-ZOE and composite.
It has been found that, the mode of sample preparation, curing procedures, aging, and extract preparation were contributing factors for the vast modification of the acute cytotoxic effects of the tested materials in mammalian cell cultures ,. It is thus impossible to biologically characterize the materials by a single test method alone, and their properties need to be investigated by various in-vitro and in-vivo tests in a structured approach .
| Conclusion|| |
Based on the conditions of the present study, it can be concluded that, composite is the recommended retrograde filling material and nano-ZOE is a promising sealer material in terms of biocompatibility.
Further studies are still needed to determine the biological response to the tested materials fromother different aspects like genotoxicity and mutagenicity.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]