Morphological, Histochemical, and Proteomic Analysis of the Effects of Fluoride and Amoxicillin, with Calcium and Vitamin D Supplementation, on Dental Enamel Formation.

Ameloblasts are specialized epithelial cells responsible for enamel formation, a process that requires tightly coordinated matrix secretion, proteolytic turnover, and hydroxyapatite crystal growth. Although genetically regulated, amelogenesis is susceptible to environmental modulation by agents such as fluoride and amoxicillin. Calcium and vitamin D supplementation have been proposed as potential protective factors against fluoride-induced enamel alterations. This study investigated whether combined exposure to fluoride and amoxicillin impairs ameloblast viability and enamel formation, and whether vitamin D and calcium supplementation can mitigate these effects. Using a rat incisor model, enamel structure, ameloblast apoptosis, and matrix protein dynamics were evaluated through ultrastructural analysis, histology, immunohistochemistry, TUNEL assay, and proteomic profiling. Fluoride exposure, particularly when combined with amoxicillin, resulted in disorganization of enamel prisms and increased apoptotic activity in ameloblasts, as evidenced by caspase-3 and TUNEL labeling, especially during the early maturation stage. Immunohistochemistry revealed reduced KLK4 levels within ameloblasts, whereas proteomic analysis demonstrated increased KLK4 abundance in the enamel matrix, suggesting altered intracellular trafficking and impaired proteolytic turnover. Additionally, the abundance of AMBN, AMTN, and ODAM, proteins essential for enamel crystal nucleation and maturation, was reduced in treated groups. Vitamin D and calcium supplementation neither significantly restored enamel organization nor normalized matrix protein alterations under the experimental conditions tested. Overall, these results indicate that fluoride exposure, particularly when combined with amoxicillin administration, disrupts ameloblast homeostasis, matrix proteolysis, and enamel prism organization, resulting in structurally compromised enamel, with no beneficial effects from vitamin D and calcium supplementation.