title: Ultrastructure of early amelogenesis in wild-type,Amelx-/-, andEnam-/-mice: enamel ribbon initiation on dentin mineral and ribbon orientation by ameloblasts.
abstract: BACKGROUND
  Dental enamel is comprised of highly organized, oriented apatite crystals, but how they form is unclear.
  
  
  METHODS
  We used focused ion beam (FIB) scanning electron microscopy (SEM) to investigate early enamel formation in 7-week-old incisors from wild-type,Amelx-/-, andEnam-/-C56BL/6 mice. FIB surface imaging scans thicker samples so that the thin enamel ribbons do not pass as readily out of the plane of section, and generates serial images by a mill and view approach for computerized tomography.
  
  
  RESULTS
  We demonstrate that wild-type enamel ribbons initiate on dentin mineral on the sides and tips of mineralized collagen fibers, and extend in clusters from dentin to the ameloblast membrane. The clustering suggested that groups of enamel ribbons were initiated and then extended by finger-like membrane processes as they retracted back into the ameloblast distal membrane. These findings support the conclusions that no organic nucleator is necessary for enamel ribbon initiation (although no ribbons form in theEnam-/-mice), and that enamel ribbons elongate along the ameloblast membrane and orient in the direction of its retrograde movement. Tomographic reconstruction videos revealed a complex of ameloblast membrane processes and invaginations associated with intercellular junctions proximal to the mineralization front and also highlighted interproximal extracellular enamel matrix accumulations proximal to the interrod growth sites, which we propose are important for expanding the interrod matrix and extending interrod enamel ribbons.Amelx-/-mice produce oriented enamel ribbons, but the ribbons fuse into fan-like structures. The matrix does not expand sufficiently to support formation of the Tomes process or establish rod and interrod organization.
  
  
  CONCLUSIONS
  Amelogenin does not directly nucleate, shape, or orient enamel ribbons, but separates and supports the enamel ribbons, and expands the enamel matrix to accommodate continued ribbon elongation, retrograde ameloblast movement, and rod/interrod organization.
authors.affiliation: Department of Biologic and Materials SciencesUniversity of Michigan School of Dentistry1210 Eisenhower PlaceAnn ArborMichigan48108; Facility for Electron Microscopy ResearchDepartment of Anatomy and Cell BiologyFaculty of DentistryMcGill UniversityMontrealQuebecH3A 0C7Canada.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Smith
authors.affiliation: Department of Biologic and Materials Sciences University of Michigan School of Dentistry 1210 Eisenhower Place Ann Arbor Michigan 48108.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Department of Biologic and Materials Sciences University of Michigan School of Dentistry 1210 Eisenhower Place Ann Arbor Michigan 48108.
authors.forename: Jan C-C
authors.initials: JC
authors.lastname: Hu
authors.affiliation: Department of Biologic and Materials Sciences University of Michigan School of Dentistry 1210 Eisenhower Place Ann Arbor Michigan 48108.
authors.forename: James P
authors.initials: JP
authors.lastname: Simmer
keywords: Ameloblast; amelogenesis imperfecta; amelogenin; enamelin; focused ion beam microscopy