ZEBRAFISH AS A MODEL IN DENTAL APPLICATIONS. A REVIEW

Headnote

Abstract.

Zebrafish (Danio rerio) are a popular model organism for scientific research, due to its small size, rapid development, and transparent embryos. One of the areas of study involving zebrafish is their teeth, which are continuously replaced throughout their lives. They have two types of teeth: incisors and pharyngeal teeth, compared to humans, who have only one type. Zebrafish teeth are replaced continuously throughout life, a process known as polyphyodonty, in contrast to humans, who are diphyodont and replace their teeth only once. Zebrafish teeth have been the subject of numerous experiments, including studies of tooth development, regeneration and evolution. In conclusion, zebrafish teeth are a fascinating subject of study in scientific research. In this review, we will describe zebrafish teeth, the experiments performed on them, and provide a comparative analysis with human teeth.

Keywords: human teeth, zebrafish teeth, model organism, pharyngeal teeth.

1. INTRODUCTION

Zebrafish are a popular model for biomedical research, playing a crucial role in studying various human diseases and biological processes, as well as human diseases, including type 2 diabetes, dyslipidemia, liver disease, neuronal disorders, cancer, infectious disease, cardiovascular disease, kidney disease, diabetes, blindness, and deafness [1].

Furthermore, zebrafish dentition offers a unique perspective for understanding tooth development, structure, and replacement mechanisms in vertebrates. Dentition of zebrafish reflects the primitive conditions found in jawed vertebrates, making them a valuable model for comparative studies [2,3]. Zebrafish possess a distinct dental pattern with three rows of teeth on each side, all of which being replaced continuously throughout their lives. This includes a ventral row of five teeth, a mediodorsal row of four teeth, and a dorsal row of two teeth, showcasing a dynamic process of tooth regeneration [3].

In dentistry, zebrafish teeth serve as an intriguing model for repetitive epithelial morphogenesis, shedding light on genetic, molecular, and developmental research [3]. Unlike human teeth, zebrafish teeth are not oral, being attached only to the fifth branchial arch. The replacement of zebrafish teeth starts early in their development, with the first tooth bud appearing at 2 days post-fertilization. Zebrafish have three rows of teeth on each side, all of which being replaced throughout life. The first tooth bud develops 2 days after post-fertilization, in position 4V, followed by the development of teeth in other positions at later stages [3,4]. This rapid replacement process involves the development of new teeth from an epithelial outgrowth, called successional lamina [3].

Comparing zebrafish teeth to human teeth reveals significant differences in their structure and development. For instance, zebrafish lack stellate reticulum or stratum intermedium in their enamel organ, contrasting with the complex enamel organ structure in mammalian teeth. Understanding these variations provides valuable insights into the evolutionary implications of tooth development across species [3].

Overall, zebrafish dentition serves as a fascinating model system for studying tooth regeneration and evolutionary aspects of dental development. By exploring the similarities and differences between zebrafish and human teeth, researchers can uncover fundamental principles underlying tooth formation and replacement mechanisms in vertebrates.

The ability of zebrafish to produce large numbers of embryos makes them particularly useful for high-throughput drug screening, allowing researchers to test the effects of a large number of compounds in a relatively short time [5-7]. This has led to the increased use of zebrafish as a model organism to study pathological processes related to human diseases, including bone disorders and kidney disease [7,8]. Automated screening procedures that utilize high-content imaging systems and artificial intelligence-driven algorithms have been developed to rapidly acquire and analyze brightfield and fluorescent images of zebrafish embryos, allowing for faster and more accurate screening outputs [6]. These platforms have been validated for a wide range of assays, including the assessment of genetic mutations and the effects of X-ray irradiation [6]. Overall, the use of zebrafish for drug screening offers a cost-effective and efficient way to identify potential drug candidates for further development.

Zebrafish are important for studying early developmental and regenerative biology, because they can repair damaged heart muscle after various types of cardiac injury, making them a valuable model for studying human genetic disorders and diseases. Also, their genetic similarities to humans, their ability to regenerate various tissues such as the heart, brain, and fins, and the transparency of embryos, allows easy observation of cell and tissue formation [9].

Compared to rodent models, zebrafish possess several advantages in the study of vertebrate development and disease, including the optical clarity of the developing embryo, which enables live imaging at organism level [10].

In summary, zebrafish are an important model organism in biomedical research, contributing significantly to the understanding of human diseases and the development of potential treatments and therapies.

2. MATERIALS AND METHODS

2.1. Search Strategy

The current systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [11], employing several electronic databases (Science Direct, PubMed, and Google Scholar) and using the following keywords: ((zebrafish teeth [Title/Abstract]) AND (human teeth [Title/Abstract])) AND (model organism [Title/Abstract]) AND (pharyngeal teeth [Title/Abstract]). Inclusion criteria referred to studies issued until March 2024, in English, which cover the main aspects of zebrafish teeth, their comparative analysis with human teeth, and the main branches in which zebrafish have been used as a model in dentistry.

2.2. Exclusion Criteria

We applied the following exclusion criteria: (1) conference abstracts, books, book chapters, and unpublished results; (2) non-English papers; (3) reviews, systematic reviews, meta-analyses, and letters.

2.3. Data Extraction

Among the initial 281 reports collected through electronic search, 27 were omitted due to duplicated results, 61 were ruled out because of the article type, and 122 review articles were omitted and deemed irrelevant based on abstract and/or title information. Additionally, 5 were excluded because they were not written in English.

2.4. Data Synthesis

Finally, 35 articles were included in this study, as demonstrated in the diagram of the literature search and selection process (Fig. 1). It was thought that the studies would be too heterogeneous to be combined, so that a narrative synthesis was performed. The results summarized the main aspects of zebrafish teeth, their comparative analysis with human teeth, and the main branches in which zebrafish have been used as a model in dentistry.

3. RESULTS AND DISCUSSION

3.1. Zebrafish teeth

Zebrafish are small, freshwater fish that possess a remarkable set of continuous, multi row teeth serving as a valuable model system for understanding the development, regeneration, and evolution of teeth in vertebrates. Zebrafish teeth exhibit several notable features, including continuous growth. Unlike many higher vertebrates, zebrafish do not experience discrete cycles of tooth loss and replacement. Instead, new teeth grow continuously behind the functional ones, allowing for efficient, uninterrupted feeding [12,13]. Also, the morphology of zebrafish teeth reflects their predatory lifestyle, featuring sharp cusps designed to grab and tear flesh [14].

On the other hand, zebrafish teeth show impressive regenerative capabilities, making them an ideal model for investigating the basic mechanisms of tooth renewal in vertebrates [13,14].

Researchers have harnessed the unique qualities of zebrafish teeth of improving the knowledge on tooth development and regeneration. Techniques such as forward genetic screening and chemical mutagenesis are used to identify the numerous genes involved in tooth and bone formation, providing insights into the regulatory networks that govern these processes [13].

Investigations into the roles of specific molecules, such as the retinoic acid (RA) [15], have also provided critical information on how these factors influence tooth regeneration and development.

On the other hand, techniques such as the BEE-ST (spatiotemporal monitoring of bone and tooth growth) approach have enabled precise tracking of tooth and bone development, regeneration and healing in various species, including zebrafish [12].

n zebrafish, the main difference between pharyngeal and oral teeth lies in their location and function. Zebrafish have both oral and pharyngeal teeth. The oral teeth are located in the mouth, while the pharyngeal teeth are found in the pharyngeal jaws, which are part of the throat. The pharyngeal teeth are used for crushing and grinding food, while the oral teeth are involved in grasping and holding prey. The first tooth bud starts to develop 2 days after post fertilization (dpf) in position 4V, and the replacement of the first-generation teeth starts early [16,17]. Zebrafish teeth have been studied to understand the dynamics of tooth formation, replacement, and attachment, and they could be used to research cell dynamics in tooth replacement in either larval stages or culture slices [3,16,18]. Zebrafish have a lifelong replacing dentition of 22 pharyngeal teeth, which are continuously replaced throughout their lives. The innervation and development of the pharyngeal teeth in zebrafish have been the subject of scientific studies, with research focusing on the nerves and epithelial origin of the pharyngeal teeth [16,19,20]. A quantitative analysis has shown that the shape of the pharyngeal teeth in zebrafish varies according to their position in the pharyngeal jaws [18].

Zebrafish's dentition is fully established, and every tooth is replaced throughout its life, making it an ideal model for studying repetitive epithelial morphogenesis. The zebrafish pharyngeal teeth are arranged in three distinct tooth rows: a row with five ventral teeth, a row with four mediodorsal teeth, and a row with two dorsal teeth, symmetrically arranged on each side of the pharyngeal skeleton. The pharyngeal teeth are continuously replaced throughout zebrafish's life, a characteristic known as polyphyodonty. Replacement of zebrafish pharyngeal teeth is a dynamic process that involves the coordinated interaction of multiple epithelial layers [16,20,21]. Therefore, the main difference lies in the location and continuous replacement of the pharyngeal teeth in zebrafish, as opposed to the two sets of teeth in humans.

Pharyngeal teeth are not unique to zebrafish, being found in many other species of fish from the Cyprinidae family, such as common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella) and ide (Leuciscus idus) (Gibert et al., 2010; Pasco-Viel et al., 2010; Su et al., 2021). However, not all fish of the Cypriniformes order have pharyngeal teeth, for example, rohu (Labeo rohita) and silver carp (Hypophthalmichthys molitrix) have pharyngeal teeth but lack buccal teeth [22,23]. However, the number, shape, and arrangement of pharyngeal teeth can vary widely between different fish species. For example, some fish species have only a few pharyngeal teeth, while others have many. Additionally, the shape and size of pharyngeal teeth can vary, depending on species and feeding habits. In zebrafish, pharyngeal teeth are arranged in three distinct tooth rows, a ventral (V), a mediodorsal (MD), and a dorsal (D) one in adults, having a total of 22 teeth [18]. The pharyngeal teeth of Zebrafish lack dentition on the oral jaws and possess pharyngeal teeth only zebrafish are used for crushing and grinding food and are involved in processing various types of food, including live prey, algae, and artificial diets [24].

3.2. Comparison between human and zebrafish teeth

Zebrafish have two types of teeth: incisors and pharyngeal teeth. The incisors, located in the anterior part of the jaw, are used for grasping and tearing prey. The pharyngeal teeth, located in the throat, are used for crushing and grinding food. Zebrafish dentition consists of three rows of teeth on each side, all of which being replaced throughout life: a ventral row (V) of five teeth, a mediodorsal row (MD) of four teeth, and a dorsal row (D) of two teeth. Replacement of zebrafish teeth occurs in a specific pattern, with new teeth formed at the base of the old teeth, pushing them out as they grow [3,16,17].

Dentition of the zebrafish is continuously replaced throughout its life, therefore it is a polyphyodont organism. This is in contrast to humans, who are diphyodonts and only replace their teeth once [16]. The process of tooth replacement in zebrafish is a subject of research due to its potential implications for understanding repetitive epithelial morphogenesis and tooth development in other vertebrates [3].

Zebrafish teeth are similar to human teeth in terms of their constitution, being made up of pulp and dentine covered with a protective layer of enamel [25]. A study mentioned that zebrafish teeth differ from human teeth but have a similar organization (Fig. 2), both having tooth crowns made of dentine covered with a protective layer of enameloid [25,26]. Additionally, zebrafish and human teeth have been studied for their development and molecular pathways, providing insights into the evolution and development of teeth in vertebrates [27].

Zebrafish and humans have different patterns of tooth replacement (Table 1). Zebrafish have a lifelong replacing dentition of 22 pharyngeal teeth, each tooth being replaced every 8-12 days in juveniles [16]. In contrast, humans have only two sets of teeth that they replace once. Zebrafish, unlike humans, have teeth located in the pharynx [2].

Zebrafish's dentition is fully established, every tooth being replaced throughout its life, making it an ideal model for studying repetitive epithelial morphogenesis [3]. This continuous tooth replacement in zebrafish is a unique characteristic not found in humans.

3.3. Experiments on zebrafish with dental implications

Experiments on zebrafish have been conducted to study various aspects of their dentition, Zebrafish continuously replace their pharyngeal teeth Pharyngeal teeth are spoon-shaped, oral teeth morphology varies among species including tooth replacement, tooth cycling, and tooth attachment. Zebrafish, with their lifelong replacing dentition, have been used as a model organism to investigate tooth development and replacement. The complete dentition of the zebrafish consists of three rows of teeth on each side, all of which being replaced throughout life. Zebrafish dentition has been studied to understand the dynamics of tooth formation, replacement, and attachment. These studies have provided insights into repetitive epithelial morphogenesis, tooth cycling independent of attachment, and the dentine-bone interface in zebrafish teeth. However, specific experiments related to dental materials and their effects on zebrafish behavior or from a neural point of view were not found in the provided search results.

Zebrafish have been used to study the effects of various substances on oral health specifically related to toxicology. First of all, zebrafish is a versatile model for studying the toxicity of various substances in several areas, including developmental toxicity, reproductive toxicity and neurotoxicity [28–30]. The studies demonstrate the utility of zebrafish in determining the toxicity of chemicals, which could be extended to the evaluation of substances relevant to oral health when considering the impact of toxicants on the development and overall body function.

Another approach is to investigate craniofacial growth and development in the context of dentistry. Several studies have confirmed the suitability of zebrafish as an animal model for dental research, particularly concerning craniofacial development [31,32]. Thus, zebrafish have been widely used for genetic screening before identifying the mutations affecting craniofacial development, leading to valuable information on the genetic regulation of craniofacial morphogenesis [31]. Advantages of using zebrafish include rapid development, transparency, and the ability to handle larger numbers of individuals, compared to other common laboratory animals. These attributes allow researchers to observe craniofacial development noninvasively and monitor gene expression patterns and environmental impacts on craniofacial morphology [33].

Zebrafish have been used in several studies on developmental biology, pathological conditions, validation studies, toxicology studies, understanding human diseases, behavioral studies, genetics research, and therapeutic agent testing (Fig. 3). Thus, the utilization of zebrafish models in genetics research focusing on craniofacial development in dentistry has provided valuable insights into the genetic mechanisms underlying facial structure formation, offering a unique perspective on the genetic basis of oral health.

Also, zebrafish have emerged as a powerful tool for testing novel therapeutic agents in dentistry, enabling researchers to efficiently screen and evaluate potential treatments with relevance to oral health conditions, ultimately contributing to the development of innovative therapeutic strategies.

By leveraging zebrafish models, researchers have made significant strides in unraveling the intricate pathways involved in the evolution of various human diseases pertinent to oral health, shedding light on disease mechanisms and paving the way for targeted interventions and personalized treatment approaches.

Recently, several studies have used zebrafish as an essential tool in conducting behavioral investigations in the field of dental research, providing a versatile platform to investigate complex behaviors relevant to oral health, such as feeding patterns, social interactions and response to stimuli, thus improving understanding behavioral aspects in dentistry. The robustness of f indings from zebrafish models in dentistry research can be further reinforced through validation studies encompassing genetic analyses and behavioral assessments, ensuring the reliability and reproducibility of experimental outcomes and facilitating the translation of research findings into clinical applications.

Zebrafish models have been instrumental in elucidating the developmental processes under both normal and pathological conditions relevant to dentistry, providing valuable insights into disease progression, tissue regeneration, and potential therapeutic targets in the oral health domain.

Also, investigations into perturbations of wnt10a expression in zebrafish have offered valuable insights into its implications for dentistry research, shedding light on the role of this critical gene in craniofacial development and oral health disorders, potentially paving the way for targeted therapeutic interventions.

Cloning of BCOR ortholog in zebrafish has enabled researchers to explore the pleiotropic effects of BCOR mutations, offering a unique opportunity to investigate the impact of genetic variations on craniofacial development and oral health outcomes, thereby expanding our understanding of the molecular mechanisms underlying dental conditions.

The potential implications of dental materials on zebrafish behavior and neural development have been the subject of some research. Zebrafish have been used as a model to evaluate the effects of dental materials on their embryonic and larval development, as well as their overall toxicity and biocompatibility. For example, a study evaluated the effects of PFM (Porcelain Fused to Metal) on the embryonic and larval development of zebrafish to determine the safety of these materials [8]. Additionally, zebrafish have been preferred in dental material toxicity studies and research related to genetic and molecular factors in tooth formation and dentition [25].

Furthermore, zebrafish have been utilized to study the toxicity of dentifrices, with findings linking exposure to certain compounds to alterations in swimming behavior, impaired neural development, and changes in neurotransmitter levels [34]. These studies highlight the potential of zebrafish as a model organism for assessing the effects of dental materials on behavior and neural development.

In summary, the research suggests that zebrafish can be valuable for studying the implications of dental materials on behavior and neural development. They have been used to assess the safety, toxicity, and biocompatibility of various dental materials, providing insights into their potential effects on zebrafish embryonic and larval development, as well as their behavior and neural function.

4.CONCLUSIONS

Zebrafish are a valuable model for studying tooth development and replacement. They have a lifelong replacing dentition of 22 pharyngeal teeth, with three rows of teeth on each side that are replaced throughout life. Zebrafish teeth are continuously replaced every 8-12 days, making them a good model for testing dental products and studying the mechanisms of tooth replacement. Zebrafish's ability to replace teeth throughout life makes it an excellent model for understanding the process of tooth regeneration, which has implications for dentistry, including the development of regenerative dental therapies. In comparison to human teeth, zebrafish teeth exhibit differences in their replacement process and attachment to the surrounding bone, providing valuable insights for potential applications in human dentistry [3,16,35].

The continuous and rapid tooth replacement in zebrafish makes them a promising model for testing dental products and for studying the mechanisms of tooth replacement, with possible implications for the development of regenerative dental therapies and understanding of human tooth development and replacement. The differences between zebrafish and human teeth provide valuable insights for potential applications in human dentistry [16,35].

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Corresponding author: Alin Ciobica; e-mail: [email protected]

References

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