Backscatter Scanning Electron Microscopic Comparisons of Enamel Protein Null Mice

Backscattered electron imaging can distinguish tissues that vary in their degree of mineralization because the quantity of high-energy backscattered (reflected) electrons increases with the atomic numbers of atoms in the specimen. The more highly mineralized a tissue is, the more calcium there is to scatter electrons. Objective: To assess enamel mineralization at 1 mm increments along continuously growing mouse incisors from wild-type, Enam, Ambn, Klk4, Mmp20 and Mmp20/Klk4 double knockout mice using backscatter scanning electron microscopy. Methods: Mandibular incisors from perfused 9 week old mice were perpendicularly sectioned at eight successive one mm increments, reembedded in plastic and diamond polished. Brightness and contrast were adjusted to a standard location of lingual alveolar bone. Results: True enamel did not form in the Enam or Ambn null mice. A thin crust of mineral that readily separated from the dentino-enamel junction formed during the secretory stage along with numerous ectopic mineral nodules within the enamel organ epithelia (EOE) that increased in number and degree of mineralization during the maturation stage. The enamel layer in Enam heterozygous mice reached normal thickness in most areas, but ectopic mineral formed in the EOE and the outer enamel showed defective decussation. Klk4 null mouse enamel achieved full thickness, but the enamel layer was progressively less mineralized with depth. Klk4 heterozygous mouse enamel highly mineralized but this mineralization was delayed. In the Mmp20 and Mmp20/Klk4 double null mice, two mineral layers formed over the dentin. The deeper layer was less mineralized. Nodules formed a third mineral layer during the maturation stage in Mmp20 null incisors, which was less apparent or absent in the Mmp20/Klk4 double null mice. Conclusion: Early (secretory stage) defects cause more severe mineralization deficits and ectopic mineralization when compared with later (maturation stage) defects. This study was supported by NIDCR grants DE011301 and DE019775.