Objective: The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment.
Materials and methods: After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique.
Results: With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis.
Conclusions: Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis.
Keywords: Dental fluorosis; Fluoride; Larva; Zebrafish.
Zebrafish culture and fluoride treatment
The adult AB zebrafish were maintained at the zebrafish facility at 28 °C with a cycle of 14 h light and 10 h dark, and they were feed with brine shrimp three times a day. One male and one female zebrafish were paired for the spawning. All the animal experiment protocols were approved by IRB of School of Stomatology, the Fourth Military Medical University.
Once the zebrafish eggs were laid, they were cultured in the embryo medium (EM)(14.955 mM NaCl, 0.5 mM KCl, 0.985 mM CaCl2-2H2O, 0.15mMKH2PO4, 0.05
General effect of excess fluoride treatment
Excess fluoride was added to the embryo medium of zebrafish right after the zebrafish eggs were delivered. Three concentrations of fluoride did not have any obvious effects on the general growth of embryos including hatching, and in some cases, 76 ppm fluoride groups even showed an earlier hatching activity. ICP-MS analysis revealed that all the trace elements were in a lower concentration range in EM (Supplementary Table 1).
Altered calcification of zebrafish teeth with the fluoride treatment
Using the whole mount alizarin red and alcian blue staining, the
The prevalence of dental fluorosis in primary teeth is considered to be relatively low (Ruan et al., 2005; Warren, Levy, & Kanellis, 2001), which might be related with the effect of ‘placental barrier’ (Jenkins, 1978). In the plenty of animal studies of dental fluorosis, rats, mice and zebrafish in the juvenile stage are generally used (Bartlett et al., 2005, Lyaruu et al., 2013). However, there is no report about dental fluorosis model of primary teeth. In this study, zebrafish eggs were
This work was supported by National Natural Science Foundation of China (81070819, 81271116, 81470728) (X.D.).