Structure and mechanical properties of Ank/Ank mutant mouse dental tissues--an animal model for studying periodontal regeneration

Abstract

Enamel, dentine and cementum are dental tissues with distinct functional properties associated with their unique hierarchical structures. Some potential ways to repair or regenerate lost tooth structures have been revealed in our studies focused on examining teeth obtained from mice with mutations at the mouse progressive ankylosis (ank) locus. Previous studies have shown that mice with such mutations have decreased levels of extracellular inorganic pyrophosphate (PP(i)) at local sites resulting in ectopic calcification in joint areas and in formation of a significantly thicker cementum layer when compared with age-matched wild-type (WT) tissue [Ho AM, Johnson MD, Kingsley DM. Role of the mouse ank gene in control of tissue calcification and arthritis. Science 2000;289:265-70; Nociti Jr FH, Berry JE, Foster BL, Gurley KA, Kingsley DM, Takata T, et al. Cementum: a phosphate-sensitive tissue. J Dent Res 2002;81:817-21]. As a next step, to determine the quality of the cementum tissue formed in mice with a mutation in the ank gene (ank/ank), we compared the microstructure and mechanical properties of cementum and other dental tissues in mature ank/ank vs. age-matched WT mice. Backscattered scanning electron microscopy (SEM) imaging and transmission electron microscopy (TEM) analyses on mineralized tissues revealed no decrease in the extent of mineralization between ank/ank cementum vs. WT controls. Atomic-force-microscopy-based nanoindentation performed on enamel, dentine or cementum of ank/ank vs. age-matched WT molars revealed no significant difference in any of the tested tissues in terms of hardness and elastic modulus. These results indicate that the tissue quality was not compromised in ank/ank mice despite faster rate of formation and more abundant cementum when compared with age-matched WT mice. In conclusion, these data suggest that this animal model can be utilized for studies focused on defining mechanisms to promote cementum formation without loss of mechanical integrity.

Figure 1

Sample preparation for nanoindentation. Lower left mandible was extracted with all three molars, incisor, and alveolar bone intact (a) then mounted at room-temperature-cure epoxy (b). Incisor was removed by a diamond wafering saw. The internal cross-section of the first molar was then exposed by grinding (600 grit then 1500 grit SiC paper) from the mesial side until the pulp was visible. Final surface smoothing was prepared by an ultramicrotome using a diamond knife (c). Nomenclatures: E = enamel, D = dentin, C = cementum, P = periodontal ligament, and B = alveolar bone.

Figure 2

Microscopy analyses of cervical cementum in WT and ank/ank animals. Histology images similar to those reported in Nociti et al [2] revealed more than a 10 fold increase in ank/ank cementum thickness compared to the WT sample (a). A similar difference between WT and ank/ank cementum was also observed at the TEM level as shown in (b) and (c). In both (b) and (c), the delineation between dentin and cementum are highlighted by white hollow arrow in the TEM images. The distinction between dentin and cementum was recognized based on the difference in the diffraction patterns. Dentin was identified by the textured 002 Hap reflections, which was absent in cementum due to a changed Hap crystal orientation. Diffraction patterns of cementum revealed no crystallographic difference between WT and ank/ank. See Figure 1 for nomenclature.

Figure 3

SEM images of enamel, dentin, and cementum in the cervical regions in backscattered mode. In both WT (a) and ank/ank mice (b), enamel showed greater mass density than dentin and cementum, while no detectable differences were found between cementum and dentin regions in both WT and ank/ank tissues. The gray-level histogram in (c) revealed no significant differences in mass density between WT and ank/ank when the same tissues were compared. See Figure 1 for nomenclature.

Figure 4

Nanoindentation measurements were made in enamel, dentin (cervical & apical) and cementum (cervical & apical). Examples of sampling regions are shown in (a) for WT and (b) for ank/ank mutant tissues. Highlighted in the rectangular outlines are indentation profiles taken from DEJ and DCJ in the cervical regions. Dotted lines in (a) and (b) denote DCJ. The circled regions are the additional sampled regions of nanoindentation.