Maturation stage enamel malformations in Amtn and Klk4 null mice

Amelotin (AMTN) and kallikrein-4 (KLK4) are secreted proteins specialized for enamel biomineralization. We characterized enamel from wild-type, Amtn^-/-, Klk4^-/-, Amtn^+/-Klk4^+/- and Amtn^-/-Klk4^-/- mice to gain insights into AMTN and KLK4 functions during amelogenesis. All of the null mice were healthy and fertile. The mandibular incisors in Amtn^-/-, Klk4^-/- and Amtn^-/-Klk4^-/- mice were chalky-white and chipped. No abnormalities except in enamel were observed, and no significant differences were detected in enamel thickness or volume, or in rod decussation. Micro-computed tomography (μCT) maximum intensity projections localized the onset of enamel maturation in wild-type incisors distal to the first molar, but mesial to this position in Amtn^-/-, Klk4^-/- and Amtn^-/-Klk4^-/- mice, demonstrating a delay in enamel maturation in Amtn^-/- incisors. Micro-CT detected significantly reduced enamel mineral density (2.5 and 2.4 gHA/cm³) in the Klk4^-/- and Amtn^-/-Klk4^-/- mice respectively, compared with wild-type enamel (3.1 gHA/cm³). Backscatter scanning electron microscopy showed that mineral density progressively diminished with enamel depth in the Klk4^-/- and Amtn^-/-Klk4^-/- mice. The Knoop hardness of the Amtn^-/- outer enamel was significantly reduced relative to the wild-type and was not as hard as the middle or inner enamel. Klk4^-/- enamel hardness was significantly reduced at all levels, but the outer enamel was significantly harder than the inner and middle enamel. Thus the hardness patterns of the Amtn^-/- and Klk4^-/- mice were distinctly different, while the Amtn^-/-Klk4^-/- outer enamel was not as hard as in the Amtn^-/- and Klk4^-/- mice. We conclude that AMTN and KLK4 function independently, but are both necessary for proper enamel maturation.