Enamel ribbons, surface nodules, and octacalcium phosphate in C57BL/6 Amelxâ /â mice and Amelx+/â lyonization

Abstract

BackgroundAmelogenin is required for normal enamel formation and is the most abundant protein in developing enamel.MethodsAmelx+/+, Amelx+/â , and Amelxâ /â molars and incisors from C57BL/6 mice were characterized using RTâ PCR, Western blotting, dissecting and light microscopy, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), backscattered SEM (bSEM), nanohardness testing, and Xâ ray diffraction.ResultsNo amelogenin protein was detected by Western blot analyses of enamel extracts from Amelxâ /â mice. Amelxâ /â incisor enamel averaged 20.3 ± 3.3 μm in thickness, or only 1/6th that of the wild type (122.3 ± 7.9 μm). Amelxâ /â incisor enamel nanohardness was 1.6 Gpa, less than half that of wildâ type enamel (3.6 Gpa). Amelx+/â incisors and molars showed vertical banding patterns unique to each tooth. IHC detected no amelogenin in Amelxâ /â enamel and varied levels of amelogenin in Amelx+/â incisors, which correlated positively with enamel thickness, strongly supporting lyonization as the cause of the variations in enamel thickness. TEM analyses showed characteristic mineral ribbons in Amelx+/+ and Amelxâ /â enamel extending from mineralized dentin collagen to the ameloblast. The Amelxâ /â enamel ribbons were not well separated by matrix and appeared to fuse together, forming plates. Xâ ray diffraction determined that the predominant mineral in Amelxâ /â enamel is octacalcium phosphate (not calcium hydroxyapatite). Amelxâ /â ameloblasts were similar to wildâ type ameloblasts except no Tomesâ processes extended into the thin enamel. Amelxâ /â and Amelx+/â molars both showed calcified nodules on their occlusal surfaces. Histology of D5 and D11 developing molars showed nodules forming during the maturation stage.ConclusionAmelogenin forms a resorbable matrix that separates and supports, but does not shape early secretoryâ stage enamel ribbons. Amelogenin may facilitate the conversion of enamel ribbons into hydroxyapatite by inhibiting the formation of octacalcium phosphate. Amelogenin is necessary for thickening the enamel layer, which helps maintain ribbon organization and development and maintenance of the Tomesâ process.We thoroughly characterized enamel formation in amelogenin null mice and determined that the mineral covering dentin in these animals is octacalcium phosphate. The initial enamel mineral has a ribbon shape, similar to the wild type. Thus, amelogenin is not required to shape the ribbons, as is currently thought, but is required to ensure that the final mineral phase is calcium hydroxyapatite.