Article pubs.acs.org/crt
Cite This: Chem. Res. Toxicol. 2018, 31, 914−923
In Vivo Molecular Toxicity Profile of Dental Bioceramics in Embryonic Zebrafish (Danio rerio) Hardik Makkar,†,# Suresh K. Verma,‡,# Pritam Kumar Panda,§ Ealisha Jha,∥ Biswadeep Das,‡ Kaushik Mukherjee,⊥ and Mrutyunjay Suar*,†,‡ †
KIIT Technology Business Incubator and ‡School of Biotechnology, KIIT University, Bhubaneswar, Orissa 751024, India Division of Paediatric Haematology and Oncology, University of Freiburg, Freiburg 79106, Germany ∥ Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada ⊥ Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 90089, United States Chem. Res. Toxicol. 2018.31:914-923. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/23/18. For personal use only.
§
S Supporting Information *
ABSTRACT: The investigation of the biocompatibility of potential and commercially available dental material is a major challenge in dental science. This study demonstrates that the zebrafish model is a novel in vivo model for investigating the biocompatibility of dental materials. Two commercially available dental materials, mineral trioxide aggregate (MTA) and Biodentine, were assessed for their biocompatibility. The biocompatibility analysis was performed in embryonic zebrafish with the help of standard toxicity assays measuring essential parameters such as survivability and hatching. The mechanistic and comparative analysis of toxicity was performed by oxidative stress analysis by measuring ROS induction and apoptosis in zebrafish exposed to dental materials at different concentrations. The molecular investigation at the protein level was done by a computational approach using in silico molecular docking and pathway analysis. The toxicity analysis showed a significant reduction in hatching and survivability rates along with morphological malformations with an increase in the concentration of exposed materials. ROS and apoptosis assay results revealed a greater biocompatibility of Biodentine as compared to that of MTA which was concentration-dependent. In silico analysis showed the significant role of the tricalcium silicate−protein (Sod1, tp53, RUNX2B) interaction in an exhibition of toxicity. The study provides a new vision and standard in dental material sciences for assessing the biocompatibility of potential novel and commercially available dental materials.
■
materials in dentistry necessitates toxicity testing.11 The conventionally used tests for assessing the toxicity profile of endodontic materials include cell culture, implantation tests, usage tests, and animal models following standard laboratory protocols.12−15 Over time, mammalian cell culture assays have proved their proficiency for in vitro toxicity evaluation at different biological end points. These end points range from indicators of cell damage, including membrane effects, cell activity, and the proliferation rate.16 A major disadvantage of cell-culture toxicity assays is their failure to simulate in vivo situations and the noncoherent extrapolation of data when compared with those of higher animals. Animal usage tests have gained enormous relevance because the entire experimental setup is under controlled laboratory conditions. These tests permit the scientific elucidation of the biocompatibility of the dental material. A large number of controls obtainable from the animal models facilitate the superior appraisal of the variables affecting the outcome of the
INTRODUCTION Bioceramics in dentistry have gained enormous popularity as dental materials showcasing numerous clinical applications such as pulp capping agents, root-end fillings, perforation repair materials, and permanent dentin substitutes.1−3 Mineral trioxide aggregate (MTA) and Biodentine are currently the most popular bioceramics used in dentistry.4 Biodentine is a calcium silicate cement composed of mainly tricalcium silicate, zirconium oxide, and calcium carbonate. The liquid component consists of calcium chloride and hydrosoluble polymer to enhance setting.5 MTA, on the other hand, comprises tricalcium silicate, dicalcium silicate, calcium dialuminate, bismuth oxide, and calcium sulfate.6 As bioceramics used in endodontics are placed in intimate contact with the periapical tissues, their nontoxic nature and biocompatibility are imperative.7 MTA and biodentine have been reported to exihibit biocompatibility with human dental pulp stem cells (hdpsc)8, which has shown them to be a cytocompatible root-ending filling material.9 Biocompatibility may be defined as a material’s ability to have a specific functional application, eliciting an appropriate host response.10 The extensive and emerging need for biocompatible © 2018 American Chemical Society
Received: May 18, 2018 Published: July 30, 2018 914
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology
reports have shown the presence of these components in the elute through different experimental methods such as inductively coupled plasma−mass spectrometry (ICP−MS).8 After incubation, 40 mL of conditioned medium was collected from the tubes and centrifuged at 400g for 1 min. The conditioned medium was then filtered off of any remaining solid particles using a 0.2 μm syringe filter. The obtained elutes were then diluted twice (×/2) and four times (×/4), following which they were preserved for experimental purposes. The pH of the dilutions was checked and adjusted to 7.4−7.6 in order to make sure that they were compatible with the biological osmolarity of the fish. The embryos were then subjected to growth in the conditioned media containing MTA and Biodentine at different concentrations. The experimental groups were as follows:
study, providing a better understanding of the toxicity of dental materials before its usage in humans.17 The search for novel models to assess the biocompatibility of dental materials is of interest due to a need to extract diverse and well-articulated data for further understanding and correlation with human models. Various in vivo models have been established by researchers reporting the biocompatibility of dental biomaterials, among which rat and mice models are the most recognized. Though these live models have been well established in toxicological as well as genetic sciences, there are issues in the maintenance cost and the time of life cycles.18 Recently, zebrafish (Danio rerio) have emerged as a potential solution to these problems and have been shown to be a promising model for toxicity screening. Moreover, being a model for vertebrate development, the bases of embryonic development are fundamentally similar between zebrafish and mammalian embryos.19−22 Reporting the toxicity of dental materials exposed to zebrafish can be easily correlated to humans because of their genetic and physiologic similarities.23,24 This study evaluated the in vivo toxicity of MTA and Biodentine at cellular and physiologic levels using the Danio rerio model. The probable mechanism of toxicity was further elucidated with the help of bioinformatics. The report reflects the utilization of Danio rerio as a potential novel model for assessing and establishing the biocompatibility of two commercially available calcium silicate-based dental materials, namely, MTA and Biodentine. Moreover, an exploration of the detailed mechanism of material protein interactions is imperative to the articulation of strategies and the design of dental materials which are safe and fit for human use.
■
• Control group: zebrafish embryo grown in HF media. • Test group 1: zebrafish embryo grown in HF media conditioned with MTA (×/4) and Biodentine (×/4). • Test group 2: zebrafish embryo grown in HF media conditioned with MTA (×/2) and Biodentine (×/2). • Test group 3: zebrafish embryo grown in HF media conditioned with MTA (×) and Biodentine (×). Evaluation of Cytotoxicity of MTA and Biodentine on Zebrafish Embryo and Larvae. Zebrafish embryo exposure to MTA and Biodentine was performed following the protocol given by Verma et al.26 Zebrafish embryos 3−3.5 h postfertilization (hpf) were selected for the cytotoxicity assay. Twenty embryos were treated with different concentrations of 500 μL of conditioned medium from each experimental group in a 24-well plate. The control group had zebrafish embryos grown in untreated HF media. The well plates were exposed to photoperiods of 14/10 h light/dark at 28 ± 1 °C. Hatching and survivability rates were determined as the number of embryos hatched and living 96 h postfertilization, respectively. The assessment of survivability and hatching rates was performed by normalizing the control group to 100%. The treated embryos, now larvae, were also assessed for any morphological changes and compared with the control group using direct observation and stereomicroscopy. The entire experiment was performed in triplicate and repeated three times. Evaluation of Cellular Reactive Oxygen Species (ROS) in Zebrafish Following Exposure to Different Concentrations of MTA- and Biodentine-Conditioned Media. To elicit the probable mechanism of cytotoxicity caused by these bioceramics, intracellular ROS were investigated. ROS were measured using flow cytometry with 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye (Sigma-Aldrich) being a permanent ROS marker.27 Control and treated larvae were sacrificed and sonicated for the preparation of cell suspensions. Sonication was performed at an amplitude of 50 Å for 10 min. The control (96 h hatched larvae cell suspension in HF media) and experimental groups were stained with 10 μM H2DCFDA for 20 min in the dark. The stained cell suspension was washed twice with 1× phosphate-buffered saline by centrifugation to remove extra stain and subjected to flow cytometry analysis (Attune acoustic focusing cytometer, Applied Biosystems, Life Technologies). The data obtained were analyzed using facsxpress 5 (Denovo, CA) and represented as a histogram. Apoptosis assays were performed using the protocol mentioned by Asharani et al. and Verma et al.28,29 Both of the control and experimental groups of live zebrafish larvae were washed twice with HF buffer and stained with 5 μg/mL acridine orange stain (AO) for a period of 20 min. The staining of larvae was followed by washing them with HF buffer and visualizing them under the green channel of a fluorescence microscope (EVOS, ThermoScientific). The quantitative determination of green fluorescence intensity of stained larvae was done with the help of ImageJ, normalizing the background fluorescence. Furthermore, quantitative determination was done with the help of flow cytometry. For flow cytometry analysis, exposed embryos (now larvae) were sacrificed, and a single cell suspension was made by sonicating them at an amplitude of 50 Å for 10 min. The cell suspension of treated and untreated larvae was then stained by acridine orange and washed twice with 1× PBS by centrifugation to remove extra stain. After being
MATERIALS AND METHODS
Zebrafish Maintenance and Embryo Culture. Adult zebrafish (Danio rerio) were maintained in an overflow container setup (Aquaneering, USA) equilibrated with fish water containing 75 g of NaHCO3, 18 g of sea salt, and 8.4 g of CaSO4 per 1000 mL. The fish were fed with fish food containing bloodworm three times a day, following which the embryos were obtained by breeding a male and a female placed in a setup box containing a net partition in a ratio of 2:1. The procedure was strictly performed under 12 h light and 12 h dark photoperiods. Viable eggs were collected and rinsed with Holtfreter (HF)25 medium followed by their maintenance in the same medium for further experiments. The chemicals used in the preparation of buffer were procured from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Preparation of Conditioned Media and Samples. Two commercially available dental materials, Biodentine (Septodont, Saint Maurs des Fossés, France) and ProRoot White MTA (Dentsply, Tulsa Dental, Tulsa, OK) were used in this study and were mixed according to the manufacturer’s instruction. The sample for the testing was prepared according to ISO 10993-12. Briefly, 1 g of each material was dispensed at the bottom of a 50 mL centrifuge tube, evenly distributing it around the tube. The tubes were incubated for 24 h at 37 °C in 5% CO2 to ensure the complete setting of the materials. The set materials were sterilized for 20 min by exposing them to ultraviolet light. The eluates of the different materials were extracted under sterile conditions, using Holtfreter (HF) medium as an extraction vehicle. HF medium was used to rear the zebrafish embryos because of their buffer compatibility with the live system of embryos; hence, it was used for conditioning the media with test materials used for experimental assays.25 The set materials in tubes were filled with 50 mL of HF medium and kept in a humid atmosphere containing 5% CO2 for 24 h to facilitate extraction. The media containing the extract of the materials were called conditional. These extracts consist mainly of calcium silicate elutes, which are present in both materials; moreover, MTA elutes contained other soluble components in comparison to Biodentine. Previous 915
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology washed, samples were analyzed in a flow cytometer (Attune focusing cytometer, ThermoScientific, USA). Statistical Analysis. All of the experiments were performed in triplicate, and statistical analyses were performed using GraphPad Prism v6.01 (San Diego, CA). Two-way ANOVA was used to assess the significance of the difference between the groups (materials and concentrations), following which Tukey’s post hoc analysis was performed to assess the differences between individual groups. In Silico Molecular Analysis. Molecular docking studies were used to determine the interaction of the ligand and the protein to know the preferred binding orientations of the ligand that confers a minimum binding energy (generally in negative energies). In this report, we have carried out molecular docking analyses using Autodock 4.2 with tricalcium silicate as the ligand and Sod1, tp53, and RUNX2B as receptor proteins.30,31 The selection of proteins was made on the basis of their functionality. Sod1 has been reported to play a key role in ROS metabolism, and similarly tp53 is known for its active functionality in determining apoptosis. RUNX2B is known for playing an important role in osteoblast differentiation and mineralization. Because of the unavailability of the 3D structure of Sod1 from Danio rerio (Zebrafish) (UniProt ID: O73872), we have modeled the 3D structure of Sod1 using the SWISS-MODEL server.32 Furthermore, the structure was validated using a Ramachandran plot and PROCHECK33 from the Structural Analysis and Verification Server (SAVES). Similarly, in the case of tp53 (UniProt ID: P79734), MODBASE34 was used to model the protein, but in the case of RUNX2B (Runt-related transcription factor: Q4VW84), the sequence was subjected to the SWISS-MODEL server32 for structural modeling. The chemical structures were retrieved from Pubchem35 and visualized using Chimera,36 and their geometry was optimized using the Gaussian 03 program. The receptor proteins were subjected to energy minimization using the Chimera program. The parameters for the chemical structures having silicon have been set for Autodock 4.2.31 Grid dimensions were set to 40 × 40 × 40 with a spacing of 1 Å3. Lamarckian genetic algorithms (LGA) were used for grid dimensions. A genetic algorithm was used for docking runs using a population size of 150 with the maximum number of evaluations set to 2 500 000 and maximal generations. The postdocking analysis was performed using Autodock 4.2 analyze tools using conformations and clusterings and visualized using Chimera.36 Two dimensional plots were generated using Ligplot+.37 Furthermore, to elucidate the correlation effect of Sod1 functionality to RUNX2B, STRING38 analysis was performed to investigate the role of other genes in the pathway of correlation.
Figure 1. Survivability of embryonic zebrafish exposed to different concentrations of MTA and Biodentine at 72 hpf. All of the measurements were taken in triplicate, and the values were presented as the mean ± SD of three independent experiments. *P < 0.05 denotes a significant change from the control and treated embryos, respectively. The number of (*) presents the degree of significance.
Cellular Toxicity Assessment of MTA and Biodentine on Zebrafish. The mechanism of cellular toxicity exhibited by MTA and Biodentine was assessed by the evaluation of oxidative stress and cellular apoptosis in exposed zebrafish larvae. As shown in Figure 4, DCFDA fluorescence in larvae cells was found to be enhanced by increases in the concentrations of both MTA and Biodentine depicting concentration-dependent ROS generation in cells. Interestingly, the generation of ROS was higher in the case of MTA as compared to that of Biodentine at same concentrations. MTA with concentration × (bulk concentration) showed the highest generation of ROS, which was decreased in serially diluted samples ×/2 and ×/4 (p < 0.05). The ROS production by Biodentine (×, bulk concentration) was significantly less than that by MTA (× and ×/2). Figure 5 shows the fluorescence of acridine orange in different parts of the larvae exposed to different concentrations of MTA and Biodentine. As shown, the fluorescence intensity was higher in larvae treated with × concentration of MTA and was decreasing with the dilution of samples from ×/2 to ×/4 concentrations. A similar observation was found in the case of Biodentine; however, in this case larvae treated with ×, ×/2, and ×/4 were exhibiting less fluorescence than those with MTA (p < 0.05). The observation was further verified and quantified with the flow cytometry. Flow cytometry analysis (Figure 6) confirmed the microscopic data and showed a consequent concentration-dependent increase in fluorescence intensity in both cases with a significant decrease in Biodentine-treated larvae (p < 0.05). In Silico Investigation of Biodentine and MTA Interactions with Zebrafish Embryos. The in silico approach was taken to investigate the molecular interaction and variation in the interaction of MTA and Biodentine with zebrafish embryos. To explore the oxidative stress effect at the molecular level, the molecular docking of tricalcium silicate, the major component of both MTA and Biodentine, was performed with the Sod1 protein of zebrafish embryos. The regulation of Sod1 has been reported as responsible for the induction of ROS in zebrafish.39 As shown in Figure 7A and Supporting Information file SV1, the interaction of tricalcium silicate with the Sod1 protein was found with the Asn54 amino acid residue via hydrogen bonding with a bond length of 2.96 Å. Moreover, other amino acid residues such as Glu, Arg, and Gly were also found to play their roles via hydrophobic interactions. Sod1 regulation showed significant consequences in programmed cell death as determined by experimental analysis. tp53 has been
■
RESULTS Interaction of MTA and Biodentine with Zebrafish Embryos. The toxicological effects of MTA- and Biodentineconditioned media were assessed on zebrafish embryos. As shown in Figure 1, the survivability rate was found to be significantly decreased with an increase in the concentration of the exposed material. Interestingly the viability of embryos exposed to Biodentine was higher as compared to that of MTA at the same concentrations. The hatching rate determination also showed a concentration-dependent influence of both Biodentine and MTA. As compared to the control, the hatching rate was found to be significantly decreased (p < 0.05) in both Biodentine and MTA exposure (Figure 2). However, it was higher in the case of embryos exposed to Biodentine as compared to those of MTA at all concentrations (Figure 2). As shown in Figure 3, the bright field microscopic observation of larvae exposed to different concentrations of MTA and Biodentine showed significant morphological changes compared to those of the control group (p < 0.05). Larvae exposed to the MTA group showed a bending of the tail and pericardial edema at ×/2 and × concentration. However, the changes were not visualized in the Biodentine-exposed group at any concentration. 916
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology
Figure 2. Hatching rate of zebrafish larvae exposed to different concentrations of MTA and Biodentine at different hours postfertilization (hpf). (A) ×, (B) ×/2, and (C) ×/4. All of the measurements were taken in triplicate, and the values were presented as the mean ± SD of three independent experiments. *P < 0.05 denotes a significant change from the control and treated embryos, respectively. The number of (*) presents the degree of significance.
■
DISCUSSION This report showcased an in-depth in vivo and in silico analysis of the biocompatibility of two dental bioceramics, putting forward a reliable, cost-effective, and comprehensive model to assess the toxicity of dental materials. To elucidate the biocompatibility of dental material in the zebrafish model as a platform, the toxicity of MTA and Biodentine was compared. The survivability assay showed a significant difference in the viability of embryonic zebrafish with Biodentine exposure compared to that with MTA. The hatching rate was also found to be influenced more in the case of MTA exposure compared to that of Biodentine. The survivability assay results were in accordance with the cytotoxicity results displayed in the literature.40−42 Similar results have been shown by nanomaterial cytotoxicity studies of AgNP, TiO2 NP, ZnO NP, and so forth.43,44 The influence on the hatching rate and survivability can be attributed to the internalization of the exposed material present along with the conditioned-media elutes through the surface of the zebrafish embryos’ chorion followed by their interaction with metabolic proteins presents in the in vivo system. MTA elutes, being a composition of calcium silicates, aluminates, and oxides, expose other components apart from calcium silicate as compared to Biodentine. This creates abnormal metabolic activity in the embryonic cells, causing the induction of oxidative stress, apoptosis, and ultimately death.45,26 At the same concentration, because of variation in the degree of internalization of material constituents inside embryos, Biodentine showcased less toxicity compared to MTA. It is important to mention that the hatching of the embryos is also affected by the influential regulation of the hela enzyme due to material accumulation.46 The hela enzyme has been reported to play an important role in embryo hatching by digesting glycoproteins polymerized at the time of fertilization for the hardening of the chorion. It can be argued that internalized MTA and Biodentine elutes interacted with hela and influenced its functionality, thus affecting the hatching rate. A morphological analysis of hatched zebrafish larvae showed significant notochord and tail bending with MTA exposure compared to that with Biodentine, confirming the higher influence of MTA. These abnormal morphological symptoms can be also be attributed to the potential role of metabolic abnormalities created due to the internalization of MTA elutes through the surface of chorion. It is of interest that the abnormal metabolic conditions due to the influential functionality of hela enzymes created due to the interaction of the internalized exposed molecules were
Figure 3. Morphological analysis of 72 hpf zebrafish larvae exposed to different concentrations of MTA and Biodentine.
found to play a significant role in apoptosis. To elucidate the consequences at the molecular level, tp53 was docked with tricalcium silicate, and the probable interaction was investigated. As shown in Figure 7B and Supporting Information file SV2, tp53 was found to interact with tricalcium silicate via the Arg310 amino acid residue through a hydrogen bond of 2.99 Å in length. Furthermore, the effect of tricalcium silicate on the differentiation of the osteoblast and odontoblast and the homeostasis of mineralization of tissue was investigated by molecular docking of the RUNX2B protein with tricalcium silicate (Supporting Information file SV3). Figure 7C shows the protein-level interaction of tricalcium silicate with RUNX2B. As shown in Figure 7C, the Leu147 amino residue was found to interact with tricalcium silicate via hydrogen bonding with a bond length of 3.04 Å. The binding energies and H-bond distance of tricalcium silicate with Sod1, tp53, and RUNX2B can be seen in Table 1. The interaction between Sod1 and RUNX2B, as depicted in Figure 8, showed a firm correlation between the two proteins with the active involvement of other proteins and transcription factors. 917
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology
Figure 4. Flow cytometry analysis of reactive oxygen species (ROS) in zebrafish larvae exposed to different concentrations of MTA and Biodentine at 72 hpf. (A) Comparative view of fluorescence intensity as a histogram presentation. (B) Bar graph presentation of mean fluorescence intensity. All of the measurements were taken in triplicate, and the values were presented as the mean ± SD of three independent experiments. *P < 0.05 denotes a significant change from the control and treated embryos, respectively. The number of (*) presents the degree of significance.
Figure 5. Apoptosis analysis of MTA- and Biodentine-exposed zebrafish larvae at 72 hpf by acridine orange staining. (A) Fluorescence microscopy image. (B) Mean fluorescence intensity of the head region. (C) Mean fluorescence intensity of the trunk region. All of the measurements were taken in triplicate, and the values were presented as the mean ± SD of three independent experiments. *P < 0.05 denotes a significant change from the control and treated embryos, respectively. The number of (*) presents the degree of significance.
embryos.25 The speculation was checked by ROS and apoptosis analyses. ROS determination by flow cytometry showed a significant increase in MTA-exposed embryos compared to those of Biodentine (Figure 4). This increased induction of ROS
speculated to interfere with the normal metabolic processes such as oxidative stress induction and apoptosis. Previous reports have demonstrated the relation between the functionality of metabolic proteins such as he1a, Sod1, and p53 in zebrafish 918
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology
Figure 6. Flow cytometry analysis of apoptosis in zebrafish larvae exposed to different concentrations of MTA and Biodentine at 72 hpf. Single-cell suspensions of treated embryos were stained with acridine orange. (A) Comparative view of fluorescence intensity as a histogram presentation. (B) Bar graph presentation of mean fluorescence intensity. All of the measurements were taken in triplicate, and the values were presented as the mean ± SD of three independent experiments. *P < 0.05 denotes a significant change from the control and treated embryos, respectively. The number of (*) presents the degree of significance.
Figure 7. Molecular docking analysis of tricalcium silicate with (A) Sod1, (B) tp53, and (C) RUNX2B of zebrafish depicting molecular interactions with amino acid residues by Ligplot analysis. The green line presents the H-bond intensity.
nanomaterial testing,47−50 confirming that the apoptosis and
can be reasoned to be responsible for the increased levels of apoptosis, which was observed in the apoptosis determination by acridine orange staining (Figure 5). The results were in line with those previously reported in the literature about the
ROS determination in zebrafish can be a distinctive parameter for determining the biocompatibility of dental materials. 919
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology
with the help of molecular docking. Sod1 has been reported to play a key role in the regulation of oxidative stress in zebrafish, and a molecular docking analysis of tricalcium silicate with Sod1 showed the interaction at the active site with an asparagine residue by hydrogen bonding. This interaction can be attributed to the influential regulation of Sod1 with the changes at their active sites. Similarly, tp53, which plays a key role in apoptosis, was docked with tricalcium silicate to investigate their interaction. Their arginine residues were found to interact by H bonding. The Runt-related transcription factor (RUNX2) has been reported to play a central role in hard tissue mineralization and its homeostasis.51 It also plays a primary role in the differentiation of osteoblasts and odontoblasts.52−54 Tricalcium silicate was found to interact with a leucine of RUNX2B at its active site, elucidating the mechanism of its influential regulation. With reference to the docking analysis and illuminating the interaction of genes, it can be indicated that
Table 1. Binding Energy, Binding Residues, and H-Bond Distance between Tricalcium Silicate and Sod1, tp53, and RUNX2B proteins/ chemicals tricalcium silicate with Sod1 tricalcium silicate with tp53 tricalcium silicate with RUNX2B
no. in clusters conformation
binding energies (kcal/mol)
binding residues
H-bond distance (Å)
3
2
−7.14
Asn54
2.96
8
7
−6.56
Arg310
2.99
2
2
−5.82
Leu147
3.04
Biodentine and MTA have tricalcium silicate as a major constituent. In order to investigate their interaction at a molecular level, a computational approach was taken further
Figure 8. Pathway analysis of interactions between Sod1 and RUNX2B of zebrafish with the help of STITCH. 920
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology
Figure 9. Summary of the workflow.
biocompatibility of novel and commercially available dental materials.
there is an influential effect of different genes on each other’s functionality. RUNX2B was found to interact with sod1 and other metabolic genes via sod2 as depicted in Figure 8. These parameters, hence, elucidated the interaction of tricalcium-based dental material with the zebrafish model. Previous reports have mentioned similar elucidations of docking analyses with respect to toxicity in the case of bacteria,55,56 cell lines,57 and zebrafish39 models. Henceforth, it can be derived from these studies that the interpretations of these parameters can be determinants of biocompatibility. With reference to the experimental results and previous reports, it can be argued that testing the biocompatibility of a dental material can be done with the zebrafish model. The parameters of cytotoxicity can be a reliable meter for their testing. Furthermore, the use of in silico molecular docking adds to the preliminary investigation of the issue. Hence, it can be interpreted that the zebrafish model is more elaborate and encompasses multiple aspects in understanding the biocompatibility of dental materials.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00129.
■
SV1: In silico investigation of Sod1 interaction with tricalcium silicate. SV2: In silico investigation of tp53 interaction with tricalcium silicate. SV3: In silico investigation of RUN X2B interaction with tricalcium silicate. (ZIP)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected].
■
ORCID
Pritam Kumar Panda: 0000-0003-4879-2302 Mrutyunjay Suar: 0000-0002-2433-9100
CONCLUSIONS This study established the zebrafish model as a reliable in vivo model for understanding the biocompatibility of dental materials. Two commercially used dental materials, MTA and Biodentine, have been investigated for their biocompatibility through different toxicity and computational assays using embryonic zebrafish. Experimentally obtained data showed a significant difference in the biocompatibility of two materials with Biodentine are shown to be more biocompatible. A mechanistic analysis showed a significantly higher increase in ROS and apoptosis in the tested model treated with MTA compared to those with Biodentine. In silico investigation revealed the molecular interaction of the base element of materials (tricalcium silicate) to metabolic proteins such as Sod1, tp53, and RUNX2B with amino acids Asp, Arg, and Leu, respectively. The pathway analysis by STRING revealed a web of interaction between the sod1 and runx2b genes for the regulation of metabolism. Hence, with the help of these analyses, a reliable strategy was developed to investigate and compare the biocompatibility of materials used in dentistry. Thus, the study put forward a new model and standard to investigate the
Author Contributions #
These authors have contributed equally as first author.
Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Trope, M., Bunes, a L.F., and Debelian, G. (2015) Root filling materials and techniques: bioceramics a new hope? Endod. Top 32, 86− 96. (2) Debelian, G., and Trope, M. (2016) The use of premixed bioceramic materials in endodontics. G. Ital. Endod 30, 70−80. (3) Makkar, H., Verma, S. K., Panda, P. K., Pramanik, N., Jha, E., and Suar, M. (2018) Molecular insight to size and dose dependent cellular toxicity exhibited by green synthesized Bioceramic nanohybrid with Macrophages for dental application. Toxicol. Res. (Camb). Advance artice (In press). DOI: 10.1039/C8TX00112J (4) Tsai, C.-L., Ke, M.-C., Chen, Y.-H., Kuo, H.-K., Yu, H.-J., Chen, C.T., Tseng, Y.-C., Chuang, P.-C., and Wu, P.-C. (2018) Mineral trioxide aggregate affects cell viability and induces apoptosis of stem cells from human exfoliated deciduous teeth. BMC Pharmacol. Toxicol. 19, 21. 921
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology (5) Rajasekharan, S., Martens, L. C., Cauwels, R. G. E. C., and Verbeeck, R. M. H. (2014) Biodentine??? material characteristics and clinical applications: A review of the literature. Eur. Arch. Paediatr. Dent 15, 147−158. (6) Torabinejad, M., Parirokh, M., and Dummer, P. M. H. (2018) Mineral trioxide aggregate and other bioactive endodontic cements: An updated overview- Part II: Other clinical applications and complications. Int. Endod. J. 51 (3), 284−317. (7) Escobar-García, D. M., Aguirre-López, E., Méndez-González, V., and Pozos-Guillén, A. (2016) Cytotoxicity and Initial Biocompatibility of Endodontic Biomaterials (MTA and Biodentine TM) Used as RootEnd Filling Materials. BioMed Res. Int. 2016, 1. (8) Tomás-Catalá, C. J., Collado-González, M., García-Bernal, D., Oñate-Sánchez, R. E., Forner, L., Llena, C., Lozano, A., Moraleda, J. M., and Rodríguez-Lozano, F. J. (2018) Biocompatibility of New Pulpcapping Materials NeoMTA Plus, MTA Repair HP, and Biodentine on Human Dental Pulp Stem Cells. J. Endod 44, 126−132. (9) Solanki, N. P., Venkappa, K. K., and Shah, N. C. (2018) Biocompatibility and sealing ability of mineral trioxide aggregate and biodentine as root-end filling material: A systematic review. J. Conserv. Dent. 21, 10−15. (10) Donaruma, L. G. (1988) Definitions in biomaterials, D. F. Williams, Ed., Elsevier, Amsterdam, 1987, 72 pp. J. Polym. Sci., Polym. Lett. Ed. 26, 414. (11) Schmalz, G. (1994) Use of cell cultures for toxicity testing of dental materials-advantages and limitations. J. Dent. 22, S6−S11. (12) Attik, G. N., Villat, C., Hallay, F., Pradelle-Plasse, N., Bonnet, H., Moreau, K., Colon, P., and Grosgogeat, B. (2014) In vitro biocompatibility of a dentine substitute cement on human MG63 osteoblasts cells: BiodentineTM versus MTA®. Int. Endod. J. 47, 1133− 1141. (13) Lim, E.-S., Park, Y.-B., Kwon, Y.-S., Shon, W.-J., Lee, K.-W., and Min, K.-S. (2015) Physical properties and biocompatibility of an injectable calcium-silicate-based root canal sealer: in vitro and in vivo study. BMC Oral Health 15, 129. (14) Schembri Wismayer, P., Lung, C. Y. K., Rappa, F., Cappello, F., and Camilleri, J. (2016) Assessment of the interaction of Portland cement-based materials with blood and tissue fluids using an animal model. Sci. Rep. 6, 34547. (15) Nasajpour, A., Ansari, S., Rinoldi, C., Rad, A. S., Aghaloo, T., Shin, S. R., Mishra, Y. K., Adelung, R., Swieszkowski, W., Annabi, N., Khademhosseini, A., Moshaverinia, A., and Tamayol, A. (2018) A Multifunctional Polymeric Periodontal Membrane with Osteogenic and Antibacterial Characteristics. Adv. Funct. Mater. 28 (3), 1703437. (16) Cook, J. A., and Mitchell, J. B. (1989) Viability measurements in mammalian cell systems. Anal. Biochem. 179, 1−7. (17) Browne, R. M. (1994) Animal tests for biocompatibility of dental materials-relevance, advantages and limitations. J. Dent. 22, S21−S24. (18) Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., Lilly, P., Sanders, J., Sipes, G., Bracken, W., Dorato, M., Van Deun, K., Smith, P., Berger, B., and Heller, A. (2000) Concordance of the Toxicity of Pharmaceuticals in Humans and in Animals. Regul. Toxicol. Pharmacol. 32, 56−67. (19) Hill, A., Jones, M., Dodd, A., and Diekmann, H. (2011) A review of developmental toxicity screening using zebrafish larvae. Toxicol. Lett. 205, S115. (20) Lieschke, G. J., and Currie, P. D. (2007) Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet. 8, 353−367. (21) Sipes, N. S., Padilla, S., and Knudsen, T. B. (2011) Zebrafish-As an integrative model for twenty-first century toxicity testing. Birth Defects Res., Part C 93, 256−267. (22) Hill, A. J., Teraoka, H., Heideman, W., and Peterson, R. E. (2005) Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol. Sci. 86, 6−19. (23) Clemens, D. M., Németh-Cahalan, K. L., Trinh, L., Zhang, T., Schilling, T. F., and Hall, J. E. (2013) In vivo analysis of aquaporin 0 function in zebrafish: Permeability regulation is required for lens transparency. Invest. Ophthalmol. Visual Sci. 54, 5136−5143.
(24) Teng, Y., Xie, X., Walker, S., Saxena, M., Kozlowski, D. J., Mumm, J. S., and Cowell, J. K. (2011) Loss of zebrafish lgi1b leads to hydrocephalus and sensitization to pentylenetetrazol induced seizurelike behavior. PLoS One 6 (9), e24596. (25) Verma, S. K., Jha, E., Panda, P. K., Mishra, A., Thirumurugan, A., Das, B., Parashar, S. K. S., and Suar, M. (2018) Rapid novel facile biosynthesized silver nanoparticles from bacterial release induce biogenicity and concentration dependent in vivo cytotoxicity with embryonic Zebrafish-A mechanistic insight. Toxicol. Sci. 161, 125−138. (26) Verma, S. K., Panda, P. K., Jha, E., Suar, M., and Parashar, S. K. S. (2017) Altered physiochemical properties in industrially synthesized ZnO nanoparticles regulate oxidative stress; Induce in vivo cytotoxicity in embryonic zebrafish by apoptosis. Sci. Rep. 7 (1), 13909. (27) Chen, X., Zhong, Z., Xu, Z., Chen, L., and Wang, Y. (2010) 2’,7’Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radical Res. 44, 587−604. (28) Asharani, P. V., Lian Wu, Y., Gong, Z., and Valiyaveettil, S. (2008) Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19, 255102. (29) Kumari, P., Panda, P. K., Jha, E., Kumari, K., Nisha, K., Mallick, M. A., and Verma, S. K. (2017) Mechanistic insight to ROS and Apoptosis regulated cytotoxicity inferred by Green synthesized CuO nanoparticles from Calotropis gigantea to Embryonic Zebrafish. Sci. Rep. 7, 16284. (30) Trott, O., and Olson, A. (2009) AutoDock Vina: inproving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455−461. (31) Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., and Olson, A. J. (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639−1662. (32) Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 31, 3381−3385. (33) Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283−291. (34) Pieper, U., Webb, B. M., Dong, G. Q., Schneidman-Duhovny, D., Fan, H., Kim, S. J., Khuri, N., Spill, Y. G., Weinkam, P., Hammel, M., Tainer, J. A., Nilges, M., and Sali, A. (2014) ModBase, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 42, D336−46. (35) Wang, Y., Xiao, J., Suzek, T. O., Zhang, J., Wang, J., Zhou, Z., Han, L., Karapetyan, K., Dracheva, S., Shoemaker, B. A., Bolton, E., Gindulyte, A., and Bryant, S. H. (2012) PubChem’s BioAssay database. Nucleic Acids Res. 40, D400−D412. (36) Meng, E. C., Pettersen, E. F., Couch, G. S., Huang, C. C., and Ferrin, T. E. (2006) Tools for integrated sequence-structure analysis with UCSF Chimera. BMC Bioinf. 7, 339. (37) Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) Ligplot - a Program To Generate Schematic Diagrams of Protein Ligand Interactions. Protein Eng., Des. Sel. 8, 127−134. (38) Franceschini, A., Szklarczyk, D., Frankild, S., Kuhn, M., Simonovic, M., Roth, A., Lin, J., Minguez, P., Bork, P., Von Mering, C., and Jensen, L. J. (2013) STRING v9.1: Protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808−15. (39) Verma, S. K., Jha, E., Kumar Panda, P., Mishra, A., Thirumurugan, A., Das, B., Parashar, S., and Suar, M. (2018) Rapid novel facile biosynthesized Silver nanoparticles from Bacterial release induce biogenicity and concentration dependent in vivo cytotoxicity with embryonic Zebrafish - A mechanistic insight. Toxicol. Sci. 161 (1), 125−138. (40) Pérard, M., Le Clerc, J., Meary, F., Pérez, F., Tricot-Doleux, S., and Pellen-Mussi, P. (2013) Spheroid model study comparing the biocompatibility of Biodentine and MTA. J. Mater. Sci.: Mater. Med. 24, 1527−1534. 922
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
Article
Chemical Research in Toxicology (41) Daltoé, M. O., Paula-Silva, F. W. G., Faccioli, L. H., GatónHernández, P. M., De Rossi, A., and Bezerra Silva, L. A. (2016) Expression of Mineralization Markers during Pulp Response to Biodentine and Mineral Trioxide Aggregate. J. Endod 42, 596−603. (42) Alsubait, S. A., Al-Fadda, S. S., Ababtain, R. A., Alghofaily, M. M., Abuelreich, S., Anil, S., Aldahmash, A., and Mahmood, A. (2016) Biocompatibility and Mineralization Potential of ProRoot Mineral Trioxide Aggregate and Biodentine on Mesenchymal Stem Cells. J. Biomater. Tissue Eng. 6, 323−328. (43) Verma, S. K., Jha, E., Panda, P. K., Thirumurugan, A., Patro, S., Parashar, S. K. S., and Suar, M. (2018) Molecular insights to alkaline based bio-fabrication of silver nanoparticles for inverse cytotoxicity and enhanced antibacterial activity. Mater. Sci. Eng., C 92, 807−818. (44) Verma, S. K., Jha, E., Panda, P. K., Mukherjee, M., Thirumurugan, A., Makkar, H., Das, B., Parashar, S. K. S., and Suar, M. (2018) Mechanistic insight into ROS and neutral lipid alteration induced toxicity in the human model with fins (Danio rerio) by industrially synthesized titanium dioxide nanoparticles. Toxicol. Res. (Cambridge, U. K.) 7, 244−257. (45) Zhu, X., Wang, J., Zhang, X., Chang, Y., and Chen, Y. (2009) The impact of ZnO nanoparticle aggregates on the embryonic development of zebrafish (Danio rerio). Nanotechnology 20, 195103. (46) Armant, O., Gombeau, K., El Houdigui, S. M., Floriani, M., Camilleri, V., Cavalie, I., and Adam-Guillermin, C. (2017) Zebrafish exposure to environmentally relevant concentration of depleted uranium impairs progeny development at the molecular & histological levels. PLoS One 12 (5), e0177932. (47) Duan, J., Yu, Y., Li, Y., Yu, Y., and Sun, Z. (2013) Cardiovascular toxicity evaluation of silica nanoparticles in endothelial cells and zebrafish model. Biomaterials 34, 5853−5862. (48) Zhao, X., Ren, X., Zhu, R., Luo, Z., and Ren, B. (2016) Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria-mediated apoptosis in zebrafish embryos. Aquat. Toxicol. 180, 56−70. (49) Ganesan, S., Anaimalai Thirumurthi, N., Raghunath, A., Vijayakumar, S., and Perumal, E. (2016) Acute and sub-lethal exposure to copper oxide nanoparticles causes oxidative stress and teratogenicity in zebrafish embryos. J. Appl. Toxicol. 36, 554−567. (50) Fang, Q., Shi, X., Zhang, L., Wang, Q., Wang, X., Guo, Y., and Zhou, B. (2015) Effect of titanium dioxide nanoparticles on the bioavailability, metabolism, and toxicity of pentachlorophenol in zebrafish larvae. J. Hazard. Mater. 283, 897−904. (51) Gaikwad, J. S., Cavender, A., and D’Souza, R. N. (2001) Identification of tooth-specific downstream targets of Runx2. Gene 279, 91−97. (52) Fisher, S., and Franz-Odendaal, T. (2012) Evolution of the bone gene regulatory network. Curr. Opin. Genet. Dev. 22, 390−397. (53) Helder, M. N., Bronckers, A. L. J. J., and Wöltgens, J. H. M. (1993) Dissimilar Expression Patterns for the Extracellular Matrix Proteins Osteopontin (OPN) and Collagen Type I in Dental Tissues and Alveolar Bone of the Neonatal Rat. Matrix 13, 415−425. (54) Linde, A., and Goldberg, M. (1993) Dentinogenesis. Crit. Rev. Oral Biol. Med. 4, 679−728. (55) Verma, S. K., Jha, E., Sahoo, B., Panda, P. K., Thirumurugan, A., Parashar, S. K. S., and Suar, M. (2017) Mechanistic insight into the rapid one-step facile biofabrication of antibacterial silver nanoparticles from bacterial release and their biogenicity and concentrationdependent in vitro cytotoxicity to colon cells. RSC Adv. 7, 40034− 40045. (56) Verma, S. K., Jha, E., Panda, P. K., Das, J. K., Thirumurugan, A., Suar, M., and Parashar, S. (2017) Molecular aspects of core-shell intrinsic defect induced enhanced antibacterial activity of ZnO nanocrystals. Nanomedicine 13, 43−68. (57) Verma, S. K., Jha, E., Panda, P. K., Thirumurugan, A., Parashar, S. K. S., Patro, S., and Suar, M. (2018) Mechanistic Insight into SizeDependent Enhanced Cytotoxicity of Industrial Antibacterial Titanium Oxide Nanoparticles on Colon Cells Because of Reactive Oxygen Species Quenching and Neutral Lipid Alteration. ACS Omega 3, 1244− 1262. 923
DOI: 10.1021/acs.chemrestox.8b00129 Chem. Res. Toxicol. 2018, 31, 914−923
