title: Enamel ribbons, surface nodules, and octacalcium phosphate in C57BL/6Amelx-/-mice andAmelx+/-lyonization.
abstract: BACKGROUND
  Amelogenin is required for normal enamel formation and is the most abundant protein in developing enamel.
  
  
  METHODS
  Amelx+/+,Amelx+/-, andAmelx-/-molars and incisors from C57BL/6 mice were characterized using RT-PCR, Western blotting, dissecting and light microscopy, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), backscattered SEM (bSEM), nanohardness testing, and X-ray diffraction.
  
  
  RESULTS
  No amelogenin protein was detected by Western blot analyses of enamel extracts fromAmelx-/-mice.Amelx-/-incisor enamel averaged 20.3 ± 3.3μm in thickness, or only 1/6th that of the wild type (122.3 ± 7.9μm).Amelx-/-incisor enamel nanohardness was 1.6 Gpa, less than half that of wild-type enamel (3.6 Gpa).Amelx+/-incisors and molars showed vertical banding patterns unique to each tooth. IHC detected no amelogenin inAmelx-/-enamel and varied levels of amelogenin inAmelx+/-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 inAmelx+/+andAmelx-/-enamel extending from mineralized dentin collagen to the ameloblast. TheAmelx-/-enamel ribbons were not well separated by matrix and appeared to fuse together, forming plates. X-ray diffraction determined that the predominant mineral inAmelx-/-enamel is octacalcium phosphate (not calcium hydroxyapatite).Amelx-/-ameloblasts were similar to wild-type ameloblasts except no Tomes' processes extended into the thin enamel.Amelx-/-andAmelx+/-molars both showed calcified nodules on their occlusal surfaces. Histology of D5 and D11 developing molars showed nodules forming during the maturation stage.
  
  
  CONCLUSIONS
  Amelogenin forms a resorbable matrix that separates and supports, but does not shape early secretory-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 Tomes' process.
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 SciencesUniversity of Michigan School of Dentistry1210Eisenhower PlaceAnn ArborMichigan48108; Facility for Electron Microscopy ResearchDepartment of Anatomy and Cell BiologyFaculty of DentistryMcGill UniversityMontrealQuebecH3A 2B2Canada.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Smith
authors.affiliation: Advanced Photon Source Argonne National Laboratory 9700 S. Cass Ave Building 431-B005 Argonne Illinois 60439.
authors.forename: Zhonghou
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Department of Biologic and Materials Sciences University of Michigan School of Dentistry 1210 Eisenhower Place Ann Arbor Michigan 48108.
authors.forename: Lorenza A-J
authors.initials: LA
authors.lastname: Donnelly
authors.affiliation: Department of Biologic and Materials SciencesUniversity of Michigan School of Dentistry1210Eisenhower PlaceAnn ArborMichigan48108; Department of Pediatric DentistrySchool and Hospital of StomatologyPeking University22 South AvenueZhongguancun Haidian DistrictBeijing100081China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
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; amorphous calcium phosphate; enamel; incisor; molar; octacalcium phosphate