Zebrafish larvae as a model for investigating dual effects of fluoride on bone development.

Fluorine is the most active non-metallic element in nature and can combine with a variety of elements to generate fluoride in the environment (Kono, 1994; Taher et al., 2024). It has been reported that when people intake excessive fluoride through such routes as air, water and soil, they will exhibit pathological conditions such as dental fluorosis, skeletal fluorosis and fluoride toxicity (Wu et al., 2022). In the regions of Africa and Asia endemic fluorosis have been accounted in the majority of the region affecting approximately 100 million people (Srivastava and Flora, 2020). Significant evidence suggests that excessive fluoride exposure can disrupt the bone turnover process, leading to an imbalance between osteogenesis and bone resorption (Wei et al., 2019; Li et al., 2022). Skeletal fluorosis is one of the main manifestations of fluoride-induced bone impairment, profoundly affecting human health. Patients with skeletal fluorosis mostly exhibit symptoms including bone sclerosis, osteochondromatosis, osteoporosis, and degenerative alterations in articular cartilage (Nair et al., 2011; Qiao et al., 2021). Numerous studies have shown that fluoride can modulate the activity of osteoblasts and osteoclasts through various signaling pathways such as PI3K/AKT, Wnt/B-catenin and PPARy (Wang et al., 2020; Zou et al., 2022). Although rodents are extensively utilized in skeletal fluorosis studies, their innate limitations have, to a certain extent, slowed down the research progress and hindered the conversion of research outcomes. Due to the long reproductive cycles and high costs, it is impossible to study the genetic and cumulative effects of fluoride on the skeletal development of multiple generations of rodents within a short period of time (Rezaee et al., 2020). Consequently, research on pharmacological targets for fluoride-induced bone impairment has been slow. This underscores the necessity for an appropriate biological model that can effectively facilitate studies in this field, allowing for quicker and more cost-effective research advancements.

Zebrafish have emerged as a highly beneficial model system in human disease research and drug screening owing to several advantageous characteristics (Patton et al., 2021). Their tiny size, ease of culture, robust reproductive potential, and rapid embryonic development make them a unique vertebrate model (Erlebacher et al., 1995; Fleming et al., 2005; Li et al., 2009; Han et al., 2015). These traits allow researchers to perform large-scale, high-throughput screening more efficiently compared to traditional animal models. During early developmental stages, zebrafish exhibit a transparent phenotype, enabling the clear visualization of internal organ structures under a microscope (Corrales et al., 2014; Burns et al., 2015; Jiang et al., 2023; Reis Santos et al., 2024). This transparency is particularly advantageous for observing and analyzing skeletal development. Furthermore, zebrafish larvae share a high degree of physiological and genetic homology with mammals in skeletal growth and development processes (Schartl, 2014; Valenti et al., 2020). These similarities further underscore the value and advantages of zebrafish as a model organism in skeletal system research.

At present, zebrafish have been increasingly applied in multidisciplinary scientific research, including studies on bone development and metabolism. Various zebrafish models for bone diseases have been successfully established, showcasing their broad application potential in studying bone injuries. Researchers have established a glucocorticoid-induced zebrafish osteoporosis model and used this model for related drug screening studies (Bergen et al., 2019; Zhou et al., 2024). Additionally, a novel zebrafish model of osteogenesis imperfecta has been established through genetic manipulation techniques (Huang et al., 2021). Zebrafish are regarded as a mature drug screening model. Researchers have developed a variety of potential drugs using the zebrafish model to improve glucocorticoid – induced osteoporosis. For example, the combination of quercetin and rutin, as well as the chitosan – chondroitin sulfate – daidzein nanoconjugate can enhance the bone mineralization of zebrafish larvae after exposure to dexamethasone (Snega Priya et al., 2024; Haridevamuthu et al., 2025). Despite zebrafish being widely used in bone research, their application in fluoride toxicity remains underexplored. No prior studies have systematically linked fluoride dose-dependent effects to bone turnover markers in this model. Elucidating the effects of fluoride on the bone system during zebrafish early developmental stages might provide valuable insights into the toxicity of fluoride.

In this study, zebrafish larvae were used to investigate the effects of fluoride on their skeletal development during the early stages of life. The degree of mineralization bones and changes in biochemical indicators related to bone development were used to evaluate the fluoride-induced bone injury model. Our findings suggest that the zebrafish fluoride – induced bone injury model developed in this study offers a promising and effective tool for fluorosis research. This model could significantly enhance the understanding of fluorosis effects and support the development of potential therapeutic strategies.