|Year : 2022 | Volume
| Issue : 1 | Page : 46
Zebrafish as model for studies in dentistry
Amanda S C. Ohashi1, Helena R de Souza Schacher1, Christiane S Pizzato2, Mônica R M. R. Vianna3, Luciane M de Menezes4
1 PhD Students, Dental Program, School of Health and Life Sciences, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil
2 Biomedicine Student, Centro Universitário Ritter dos Reis, Porto Alegre, Brazil
3 Professor at the Postgraduate Programs in Biology Cellular and Molecular and in Ecology and Evolution of Biodiversity, ZebLab, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil
4 Professor at the School of Health Sciences and life (Dental Program) of the Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil
|Date of Submission||13-May-2022|
|Date of Decision||22-Jul-2022|
|Date of Acceptance||02-Aug-2022|
|Date of Web Publication||13-Oct-2022|
Luciane M de Menezes
Dental Program, School of Health and Life Sciences, Pontifícia Universidade Católica do Rio Grande do Sul, 6681 Ipiranga Avenue, Building n. 6, Porto Alegre, RS, 90619-900
Source of Support: None, Conflict of Interest: None
INTRODUCTION: Over the last years, zebrafish has gained prominence in the biomedical community. It is currently considered one of the best vertebrate animal models for various types of studies, such as toxicology and developmental biology.
OBJECTIVE: The aim of this study was to conduct a literature review on the use of zebrafish in dentistry and whether this animal model could be a viable alternative for performing different types of studies in this area.
METHODS: A literature search was performed using the PubMed, Lilacs, Embase, and Dentistry and Oral Sciences Source. The keywords used as search terms were zebrafish and dentistry. The selection criteria were articles published in English that used zebrafish as an animal model in dentistry, oral health, and craniofacial growth/development.
RESULTS: The electronic search of literature yielded 421 articles. After the analysis of the abstracts, 29 articles were selected for an in-depth analysis and reading of the full text.
CONCLUSIONS: All studies included in this review confirm zebrafish's excellence as an animal model for various types of dentistry studies, as well as assisting and complementing other studies involving mammals.
Keywords: Animal model, dentistry, Zebrafish
|How to cite this article:|
Ohashi AS, de Souza Schacher HR, Pizzato CS, Vianna MR, de Menezes LM. Zebrafish as model for studies in dentistry. J Orthodont Sci 2022;11:46
| Introduction|| |
Zebrafish (Danio rerio) is a freshwater fish native to Southeast Asia. The current use as a model organism occurred from the work of George Streisinger, a pioneer in the use of molecular genetics for the study of embryology of vertebrates, and Kimmel,,, who published detailed descriptions of cell differentiation and organization of the nervous system.
Although mammals are considered the gold standard for developmental toxicity assessment, zebrafish has been increasingly used for in-vivo chemical toxicity.,, It represents a viable alternative, considering their small size, high fecundity, embryo optical transparency, rapid embryonic development, and low maintenance cost. Developmental toxicity, reproductive toxicity, cardiovascular toxicity, neurodevelopmental toxicity, and ocular developmental toxicity of hazardous chemicals have been effectively studied using zebrafish model. Considering this, in 2021, a quick search in the PUBMED/NCBI database with “zebrafish” as a keyword resulted in about 42,700 articles.
Zebrafish is currently considered one of the best vertebrate models for developmental biology studies. Despite anatomical and histological differences from mammals, including the lack of organs such as lungs, prostate, and mammary glands, it maintains the general aspects of vertebrate body plan and constituents at anatomical, molecular, and physiological levels.,, In addition, zebrafish genome has homologues of 70% of human genes and more than 80% of those associated with human diseases, which allows a more direct extrapolation of findings than studies using invertebrates and supports the significant investment in this species in several areas translational biomedical research areas.
The zebrafish embryo develops rapidly, with primordia of all major organs appearing within 36 h postfertilization, and most of its organs such as brain, heart, and kidneys are functional on the 5th day of postfertilization. Sexual maturity is reached between 3 and 6 months of life.
The visualization of the embryonic development is one of the main advantages of the zebrafish, allowing the monitoring of the development of its various organs with the aid of a stereomicroscope. The relatively large size of the embryo (~650 μm vs. ~90 μm of the mouse embryo) and its transparency allow the use of less invasive real-time techniques without interventions such as surgery or postmortem examinations. Injection of transgenes into the zebrafish can be performed with injection into the cytoplasm, while in the rodent embryo this should be performed in their pronuclei, so the equipment for this type of procedure is less expensive and the technique easier to perform.
This article aimed to review the literature on the use of zebrafish in dentistry and whether this animal model can be a viable alternative for the accomplishment of various types of study.
| Material and Method|| |
An electronic review of the literature was performed using the PubMed, Lilacs, Embase, and Dentistry and Oral Sciences Source databases. The keywords used as search terms were zebrafish and dentistry. The selection criteria were articles published in English that used zebrafish as an animal model for any type of research in dentistry, oral health, and craniofacial growth/development.
[Figure 1] shows a flowchart of the article's selection process.
The selection of the articles was carried out independently by two investigators (A.H., H.S.) and double-checked by a second researcher (L.M.) when necessary. Titles and abstracts of potentially relevant articles were analyzed before the full text was obtained.
| Results|| |
The electronic search of literature yielded 421 articles. After the analysis of the abstracts, 35 articles were selected for an in-depth analysis and reading of the full text. Six articles were excluded because they did not use the zebrafish as animal model in the experiments or because they were conference papers, hypothesis, informative consortium, or commentaries. Thus, 29 articles were included in this study [Figure 1].
Of these 29 articles, 15 evaluated the expression of specific genes in the development of craniofacial and/or dental anomalies.,,,,,,,,,,,, Nine of the papers studied the pathogen mechanism of action in infectious diseases,,,,,,,,, three studies tested the toxicity of materials used in dentistry,,, and two were literature reviews related to zebrafish and the development of craniofacial malformations, especially cleft lip and/or palate.,
[Table 1] summarizes the results of articles selected from the electronic search.
|Table 1: Studies using zebrafish as a model animal for research in dentistry found in the databases searched|
Click here to view
| Discussion|| |
Biomedical research relies on model organisms to study biologic processes conserved between humans and lower vertebrates. Usually, the organism models are small mammals, like rats and mice. Meanwhile, in the past decades, zebrafish has been widely used as an experimental model, considering important advantages over other models and considering the fact that the results can be further validated in mammals. It is an appealing model for toxicological, genetic, and behavioral studies, as well as for testing new therapeutic agents, understanding the mechanism of evolution of several human diseases, and the development under normal and pathologic conditions.
Within oral health and dentistry, the vast majority of studies involve genetics and craniofacial development. Several researchers have investigated the role of specific genes in nonsyndromic clefts of the lip and palate.,,,, Other craniofacial malformations and syndromes have also been extensively studied,,,, as well as dental development disturbances.,,, Craniofacial development is a sensitive process and environmental factors may disrupt neural crest cells' formation, survival, migration, and differentiation. Chiquet et al. demonstrated that the knockdown of CRISPLD2 causes a differentiated expression of 249 genes in zebrafish. The interaction between three of these genes (CASP8, FOS, and MMP2) had a significant relationship with nonsyndromic cleft lip and palate. Leslie et al. discovered a genome-wide significant association with a missense variant in GRHL3 and replicated the result in an independent sample. In both the discovery and replication samples, it conferred an increased risk for cleft palate, concluding that the mutation is an etiologic variant for nonsyndromic orofacial clefting. Other variants were identified as functional for nonsyndromic cleft lip and/or palate, unraveling the role of some genes in the etiology of malformation.,, Due to the multifactorial nature of cleft lip and/or palate, animal models can be used to understanding the full spectrum of this human phenotype. Zebrafish mutants are invaluable in identifying novel candidate genes for this complex disease and testing environmental factors, contributing to understanding the etiology and discovering potential therapeutic remedies for this multifactorial disorder.
The use of pharmaceuticals during pregnancy can lead to craniofacial malformations and the use anti-epileptic drug and mood stabilizer called valproic acid is frequently associated with craniofacial teratogenicity. The use of this anti-epileptic can cause fetal valproate spectrum disorder, with symptoms such as intellectual disability, facial abnormalities (including cleft), and cardiac defects. Recently, Gebuijs et al. investigated the effects of valproic acid on cartilage and bone formation in the zebrafish larval head and concluded that the drug disturbed neural crest cells' function leading to defects in cartilage and bone formation. The zebrafish is an excellent model for investigating the genetic and environmental factors, as well as their interaction with craniofacial malformations.
One of the main advantages of using zebrafish to study infectious diseases is the possibility of noninvasive imaging at the cellular level; whereas larval zebrafish can be kept transparent, the fluorescently labeled microorganism after injection into zebrafish larvae can be monitored. The opportunistic pathogen Candida albicans is widely studied with the zebrafish model, considering that the understanding of the mechanism of action is essential to develop new therapeutics in infectious diseases. Chao et al. demonstrated that C. albicans could colonize zebrafish at multiple anatomical sites, causing mortality after being injected into the peritoneal cavities. Chen et al. investigated C. albicans adhesion factors and Lu et al. suggested that a strong biofilm activity of DST659 contributed to a high mortality rate in zebrafish hosts and poor renal function in patients, in the northern Taiwan. Candidalysin is a cytolytic peptide toxin secreted by C. albicans hyphae, which induced immune activation and neutrophil recruitment and promoted mortality in zebrafish and murine models of systemic fungal infection. Recent zebrafish research showed that enolase plays an important role in invasive candidiasis and also shows that CaS1 may be potentially useful for the development of immunotherapeutic agents against the infection.
Gram-positive Enterococcus faecalis, also an opportunistic pathogen, is frequently responsible for nosocomial infections and represents one of the most common bacteria isolated from root canal infections. The resistance to antibiotics and the capacity to form biofilms cause serious therapeutic problems to infections. Two recent studies isolated characterized the bacteria and tested bacteriophages with therapeutic potential., The phages SHEF 2, 4, and 5 were able to rescue zebrafish embryos from E. faecalis systemic clinical strain infection. SHEF2 was tested again and cleared a lethal infection of zebrafish when applied in the circulation. The phage described could be used to treat a broad range of antibiotic-resistant E. faecalis infections.
Porphyromonas gingivalis is a gram-negative anaerobe considered the key oral pathogen for severe periodontitis. P. gengivalis frequently enters the bloodstream at oral sites causing a transient bacteremia. In view of this, periodontitis affects the general health status and systemic inflammation. Systemic conditions such as cardiovascular disease, atherosclerosis, and diabetes have been linked to periodontitis. Widziolek et al. studied the mechanisms of systemic pathogenicity of Pg. They concluded that zebrafish is a suitable model to study both the host's response to P. gegivalis infection and bacterial virulence. Data revealed the first real-time in-vivo evidence of intracellular P. gengivalis within the endothelium and established that gingipains are linked to systemic disease and potentially contributed to cardiovascular diseases. The outer membrane vesicles produced by P. gingivalis mediated increased vascular permeability, leading to diseased phenotype both in vitro and in vivo. Gingipains presented on this membrane's surface could mediate vascular events, as a mechanism that involves proteolytic cleavage of endothelial cell–cell adhesins, mediating systemic disease. Farrugia et al. determined that P. gengivalis directly mediate vascular damage in vivo by degrading PECAM-1 and VE-cadherin.
The zebrafish is also increasingly used for assessing chemical toxicity and safety. In dentistry, Zhao et al. conducted a study where several metal alloys make up the porcelain-fused-to-metal (PFM) crowns, a restoration technique widely used in dentistry, but not yet systematically evaluated in vivo. The effects of PFM on the embryonic and larval development of zebrafish were evaluated in order to determine the safety of these materials. Gold–palladium (Au–Pd), silver–palladium (Ag–Pd), nickel–chromium (Ni–Cr), cobalt–chromium (Co–Cr), and titanium (Ti) porcelain crowns were immersed in artificial saliva for 1, 4, and 7 weeks and the leached solutions were collected and used to treat embryos from 4 to 144 hpf. The toxicity parameters evaluated were mortality, spontaneous movement, heart rate, hatchability, malformation, and exploratory behavior. One week of exposure to the five alloys of PFMs was not toxic to zebrafish. Mortality and malformation rates in the Ni–Cr alloy group were increased while spontaneous movement, heart rate, and exploratory behavior were decreased at 4- and 7-week exposures. The Ni–Cr alloy was the most toxic, followed by the Co–Cr and Ag–Pd alloys. The Ti and Au–Pd alloys presented good biocompatibility and were therefore the most suitable for clinical applications.
Alifui-Segbaya et al. evaluated the toxicological and teratogenic effects of three types of methacrylates (E-Denture, E-Guard, and Dental SG) used in dentistry for three-dimensional denture bases, splint, restraints, surgical guides, and diagnostic models on initial development of zebrafish. These methacrylate samples were tested in solutions of pure water and solutions with alcohol. The results showed that biocompatibility was influenced by the physicochemical characteristics of the materials, which subsequently influenced their residual monomer content before and after ethanol treatment. Although these materials showed a significant increase in degree of conversion after immersion in ethanol, more than a twofold increase was observed in E-Guard materials. Nevertheless, all methacrylates were unsafe in zebrafish bioassays. With the increasing use of 3D printers and materials on the market, the authors defend that the decision to purchase such products is based not only on economic factors but on those that will provide long-term benefits. According to authors, zebrafish bioassay is a reliable toxicological screening tool that could add to the existing biological evaluation tests in dentistry, where a number of devices for prosthetic treatments are constructed with methacrylates. More recently, Hsieh et al. verified the feasibility of electrolyzed oxidizing water as a mouthwash through the evaluation of its in-vivo toxicity by embryonic zebrafish and antimicrobial efficacy against Streptococcus mutans. Except for the 0.2% chlorhexidine gluconate, all the hypochlorous acid specimens and 2.0% chlorhexidine gluconate revealed similar antimicrobial properties. The electrolyzed oxidizing water comprising both 0.0125% and 0.0250% hypochlorous acid showed > 99.9% antimicrobial efficacy but with little in-vivo toxicity, illuminating the possibility as an alternative mouthwash for dental and oral care.
To date, most studies conducted on zebrafish to date have concluded that the correlation between zebrafish and rodent toxicity is high, thus being a viable alternative to toxicity tests in rodents and other animals.
| Conclusions|| |
This article was designed to provide information on the use of zebrafish as a model organism for studies that require a well-developed vertebrate animal model, especially in dentistry, where this model is still rarely used. All studies included in this review confirm the zebrafish's excellence as an animal model for a variety of study types. The innumerable characteristics of this animal can be useful in the evaluation of potentially toxic substances, besides being able to aid and complement other studies involving mammals.
Authors contributions/Credit author statement
All authors contributed to the study conception and design. All authors read and approved the final manuscript.
1ASCO, 1HRSS, 2CSP: methodology, investigation, writing, and preparation.
3MRMV: conceptualization, validation, writing – review and editing, and supervision.
4LMM: conceptualization, validation, writing – review and editing, and supervision.
This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior—Brasil (CAPES). The authors declare no conflict of interests. MRMV is a CNPq fellowship recipient.
Financial support and sponsorship
This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior—Brasil (CAPES).
Conflicts of interest
There are no conflicts of interest.
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