Amelogenesis

Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologically aligned, parallel, ∼50 nm wide, microns-long nanocrystals, bundled either into 5-μm-wide rods or their space-filling interrod. The orientation of enamel crystals, however, is poorly understood. Here we show that the crystalline c-axes are homogenously oriented in interrod crystals across most of the enamel layer thickness. Within each rod crystals are not co-oriented with one another or with the long axis of the rod, as previously assumed: the c-axes of adjacent nanocrystals are most frequently mis-oriented by 1°-30°, and this orientation within each rod gradually changes, with an overall angle spread that is never zero, but varies between 30°-90° within one rod. Molecular dynamics simulations demonstrate that the observed mis-orientations of adjacent crystals induce crack deflection. This toughening mechanism contributes to the unique resilience of enamel, which lasts a lifetime under extreme physical and chemical challenges.

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes may contribute to caries. For the present study, we used DNA samples collected from 1,831 individuals from various population data sets. Single nucleotide polymorphism markers were genotyped in selected genes (ameloblastin, amelogenin, enamelin, tuftelin, and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Associations with caries experience can be detected but they are not necessarily replicated in all population groups and the most expressive results was for a marker in AMELX (p=0.0007). To help interpret these results, we evaluated if enamel microhardness changes under simulated cariogenic challenges are associated with genetic variations in these same genes. After creating an artificial caries lesion, associations could be seen between genetic variation in TUFT1 (p=0.006) and TUIP11 (p=0.0006) with enamel microhardness. Our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Dental enamel formation depends on tightly regulated mineral deposition and organic matrix removal during the maturation stage. Fluoride (F) adversely affects the maturation stage of amelogenesis, causing hypomineralized enamel defects. However, hypomineralized enamel defects have increased worldwide in recent years, making it urgent to understand how co-exposure to other environmental contaminants affects fluorosis and maturation-stage amelogenesis at fluoride levels sufficient to induce defects during early-life exposure conditions in experimental models. This study aimed to determine whether lead (Pb) co-exposure amplifies fluoride-induced disturbances in the enamel maturation stage by mapping depth-dependent changes in mineral, organic, and water components across the enamel layer. Pregnant Wistar rats and their offspring were exposed to F (50 ppm), Pb (30 ppm), or both in drinking water from gestation to postnatal day 30. Mandibular third molars were evaluated using a modified Thylstrup–Fejerskov index and analyzed by quantitative microradiography and polarized light microscopy to obtain depth-resolved compositional data. Mean defect scores were 0.0 (Control), 2.6 (F), and 3.2 (Pb + F). Entire enamel thickness mineral volume decreased from 71.0% (Control) to 41.1% (F) and to 35.2% (Pb + F), accompanied by a marked increase in organic matrix volume (9.3% to > 30%) (p < 0.0001). Spatial analyses showed minimal disturbances and smaller effect sizes at the superficial region, with progressively larger effects toward the central and inner enamel regions. These findings demonstrate that Pb co-exposure exacerbates fluoride-induced enamel hypomineralization and disrupts the normal spatial pattern of enamel maturation, emphasizing the importance of combined environmental exposures for understanding mechanisms governing the development and maturation of mineralized tissues.