As already discussed, in a dentin maturation study it is desirable to analyze the whole of dentin, from predentin to dentinoenamel junction, in many stages of
development and interpret results accordingly. Imaging analysis techniques applied on a suitable developing dentin model have the potential to gather important
information on the subject. The objective of this dissertation is to validate a fetal bovine model for the study of changes in dentin mineral and matrix during
maturation using spectroscopic imaging analysis, describe these changes in quantity and quality terms and apply the same approach to evaluate developing murine dentin. Four topics are presented: topographical representation of mantle and circumpulpal dentin mineral properties of the developing bovine incisor, studies of mineral changes during dentin maturation of the bovine incisor, studies of matrix changes during dentin maturation of the bovine incisor and studies of mineral properties changes of the developing mouse molar. The specific aims in this thesis and the chapters in which the relevant experiments are described are listed below: Specific Aim I: To test the hypothesis that spatial variation in dentin mineral and matrix properties during maturation is a function of both tissue age and the tissue’s histological variation and that a fetal bovine incisor incisor model can be used for the study of dentin maturation. This specific aim is addressed in Chapter II, which describes the evaluation of two mineral properties, mineral: matrix ratio and -on a semi- quantitative basis- crystallinity, through images of spatial distribution for the two properties. Analysis of the two main dentin compartments, mantle and
and in control 1-1.5yr-old bovine incisors was conducted. Preparation of the
samples, the FTIRI system, spectroscopic parameters and the imaging techniques used are described in the Methods section. Patterns of spatial variation that mineral properties present in this section are examined and whether they represent different tissue ages or an inherent variation in the dentin tissue properties is discussed.
Specific Aim II: To describe the changes in mineral properties during mantle and circumpulpal dentin maturation and compare them between the two dentin regions. This was accomplished in the study described in Chapter III, where quantitative results for dentin mineral during maturation in mantle and in circumpulpal dentin are presented. A different FTIRI system and processing techniques were used in this part and are described. Groups of spectra, from a particular development stage of mantle or circumpulpal dentin, were extracted from the imaging files and processed for analysis. Changes in the apatite crystallinity, the carbonate and the acid
phosphate substitution in the dentin apatite are described for each maturation stage. The type of carbonate substitutions is also evaluated and the results from this part are compared to existing dentin data and similar data reported for developing bone. In the second part of Chapter III, localized by microdissection mantle and
circumpulpal dentin samples of consecutive tissue ages were acquired for FTIR spectroscopical analysis of the maturing matrix.
Specific Aim III: To test the hypothesis that there is a substantial change in relative content of highly phosphorylated proteins or decrease in the level of phosphorylation of phosphorylated proteins in dentin matrix during maturation and that this change is different in mantle and circumpulpal dentin, as reflected in their
relative content of phosphoproteins. The experiment addressing this specific aim is described in Chapter IV. A whole matrix amino acid and matrix phosphorylation analysis was performed on, similar to the ones analyzed in Chapter III,
microdissected dentin specimens. Phosphorylation was examined because, as discussed above, it is believed to be the most relevant to biomineralization post- translational modification of matrix proteins and because it has been reported to vary with dentin maturation. Amino acid analysis was performed to investigate possible changes in the relative noncollagenous protein concentration. As in Chapter III, these specimens came from either mantle or circumpulpal dentin, covering a range of tissue development stages.
Specific Aim IV: To test the hypothesis that the model of mineral maturation used holds within species. In Chapter V, the feasibility of analyzing developing mouse molars with a similar approach to that used on bovine teeth in Chapters II and III was investigated. The patterns of spatial variation in mineral properties that developing mouse molars exhibit were analyzed and association of these patterns with dentin maturation and histological variability within the crown dentin is
discussed. This study was undertaken to establish a methodology for prospective studies of the effects of deletion or mutation of dentin matrix proteins on forming mineral at different developmental stages of dentin. These studies will be studies of dentin mineral from early formation phases through maturity on mice transgenic for one or more of the proteins postulated to have a function in dentin formation and mineralization.
Fig. 1-1: Structure of a tooth. (a): 3D rendering of a microcomputed
tomography analysis of a fetal bovine I4 incisor. Cervical and incisal parts are marked. (b): view on the sagittal plane as shown in a. p=pulp,
d=dentin, e=enamel. 1mm p d e cervical incisal a. b.
Fig. 1-2: Scanning electron image from a sectioned surface of a
PMMA embedded fetal bovine incisor –backscattered electron imaging mode. The surface of the incisor was etched with .5N HCL to expose dentinal tubules. (a): lower magnification. e=enamel, md=mantle dentin, cd=circumpulpal dentin, pd=predentin. Note the decrease in density and diameter of the dentinal tubules from the predentin to the mantle dentin. (b): higher magnification of insert area in a. The peritubular dentin and intertubular dentin are marked.
peritubular intertubular md e cd pd a. b.
Fig. 1-3: Representative IR spectrum of dentin. The bands representing main vibrational modes of interest are marked.
Fig. 1-4: Fourier Transform Infrared Imaging analysis. (a):principle of operation (b):the imaging system.
a.
CHAPTER II
CHARACTERIZATION OF MINERAL AND MATRIX CHANGES IN A BOVINE