The role of amelogenin during enamel-crystallite growth and organization in vivo

Abstract

Amelogenin is critical for enamel formation, and human amelogenin gene (AMELX) mutations cause hypoplastic and/or hypomaturation enamel phenotypes. The Amelx null (AKO) mouse has a severe hypoplastic phenotype. This study evaluated the effect of amelogenin loss on enamel formation and crystallite morphology. Enamel from AKO and wild-type (WT) mice was used. The AKO mice were mated with transgenic mice expressing the most abundant known amelogenin isoform, TgM180-87, to rescue (KOM180-87) the enamel crystallite phenotype. Molar enamel was embedded, sectioned with a diamond microtome, and images were obtained by transmission electron microscopy. The crystallite sizes from multiple sections were measured using Image J. The mean thicknesses (WT = 26 nm, AKO = 16 nm, and KOM180-87 = 25 nm) and the mean widths (WT = 96 nm, AKO = 59 nm, KOM180-87 = 85 nm) of crystallites were measured. Despite a complete loss of amelogenin in AKO mice, a mineralized enamel layer with well-defined and organized crystallites is formed. In the absence of amelogenin, enamel crystallites were reduced in thickness and width. For the first time we show that introduction of the m180 amelogenin isoform into the AKO mouse through cross-breeding rescues the crystallite phenotype. We conclude that amelogenin is essential for the development of normal crystallite size.

Figure 1

Enamel crystallites in the WT mice (A and B) were well-developed and often showed a parallel organization with respect to adjacent crystallites. In contrast the AKO enamel crystallites (C) were smaller and less organized than the WT crystallites. The KOM180-87 mouse (D) showed a return to normal crystallite development in both size and with an often parallel orientation similar to that seen in the WT enamel. (TEMs all exposed at 270K magnification) (scale bar = 50 nm).

Figure 2

The enamel crystallite widths measured from TEM photomicrographs were significantly reduced in the AKO mice compared with the WT mice. The KOM180-87 mice had widths that were similar to the WT mice.

Figure 3

The enamel crystallite thickness was markedly reduced in the AKO mice compared with the WT enamel. Replacement of amelogenin in the KOM180-87 mice rescued the crystallite phenotype and allowed development of crystallites with normal thicknesses.