The Development of Enamel Structure in Mammals 277 



methods which have been used are, therefore, those which would give information 

 about the shape of the mineralising front and the orientation of the crystallites with 

 respect to it. 



Materials and methods 



Material was obtained from the tooth germs of 20 mammalian species belonging 

 to 9 orders (list in Boyde, 1964). Developing enamel and ameloblasts were fixed in 

 Palade or Dalton fixatives and embedded in 2 : 1 butyl : methyl methacrylate prior 

 to ultra-thin sectioning for electron microscopy. 



Crystallite orientation was determined in electronmicrographs by estimating the 

 degree of foreshortening of sectioned crystallite fragments (It having been determined 

 that all the crystallites In enamel are extremely long), and later by examining stereo- 

 pair electron-micrographs of the same material when the orientation of the crystallite 

 fragments with respect to the plane of section could be seen directly. 



Half-micron thick sections of the same blocks, stained with crystal violet and 

 basic fuchsin to distinguish enamel from ameloblasts, were used for the preparation 

 of wax reconstructions of the mineralising front. It was thus possible to relate 

 crystallite orientation to the shape of the mineralising front directly. 



The surface of the developing enamel was also studied directly by scanning elec- 

 tron microscopy (Boyde and Stewart, 1963). 



More recently some success has been obtained with carbon replication of the sur- 

 face of developing enamel. Tooth germs were fixed In N.F.S. to allow a more perfect 

 stripping of the ameloblasts from this surface, which is then covered with a layer of 

 evaporated carbon. This layer Is to constitute the replica: It Is peculiarly liable to 

 fracture and therefore very difficult to recover because of the very angular profiles 

 which it contains. The basis of the method of recovery Is to destroy the tooth by 

 removing the hydroxyapatlte by acid decalcification and the organic matrix with 

 ethylene-diamine. It has been found most satisfactory to remove the organic com- 

 ponent first (by refluxing ethylene-diamine In a Soxhlet condensor) leaving the 

 mineral to provide some support for the carbon. The latter Is further protected by 

 being competely surrounded by calcium phosphate powder during the extraction 

 process. After washing by distilling water Instead, the contents of the Soxhlet con- 

 densor are washed into N/2 HCl when the hydroxyapatlte of the tooth and the 

 calcium phosphate powder both dissolve. The replica fragments are collected direct 

 on grids, washed In water and dried. Photogrammetric measurements have been made 

 on stereo-pair electron-micrographs of these replicas. 



Results and discussion 



The surface of developing mammalian enamels contains depressions occupied by 

 the Tomes' processes of the ameloblasts: these depressions are approximately hexa- 

 gonal in outline at their most superficial level. They do not "fill In" symmetrically. 

 One side (usually the cervical side) of each depression grows most rapidly and Is 

 more or less flat. The remaining sides constitute together a continuous curved plane 

 making a sharp angle at the base of the depression where they meet the cervical 

 side — the prism boundary forms here. 



The majority of the crystallites develop roughly perpendicular to the surface of 

 the flat cervical sides of the depressions. In the remaining cuspal and lateral sides 



