In the studies of this dissertation, the use of a fetal bovine incisor model analyzed by FTIRI for the study of mineral changes during dentin maturation was evaluated, the evolution in maturation of mantle and circumpulpal dentin as two dentin compartments distinct in mineral properties was established and changes in these mineral properties during maturation in mantle and circumpulpal dentin were described. Changes in properties of the dentin matrix during maturation were also analyzed by FTIR spectroscopy, organic phosphate and amino acid analyses. Finally, the application of the same analytical method in 6 day-mouse molars was evaluated.
In Chapters II and III, imaging analysis for mineral properties and spectral analysis of mantle and circumpulpal dentin areas of successive tissue ages for the same mineral properties was performed on dentin of fetal bovine incisors. It was shown that during dentin maturation there is an increase in mineral:matrix and crystallinity of the mineral, while the relative content of mineral in acidic phosphate decreases. This pattern holds true for both mantle and circumpulpal dentin and the biggest part of the changes described mainly occurs in early stages of maturation, while in later stages the levels for the same mineral properties reach a plateau or continue changing very gradually. Based its lower levels of mineral:matrix and
crystallinity, a mantle dentin zone could be differentiated from circumpulpal dentin early in maturation and continued to represent an approximately 80-100μm wide dentin strip next to DEJ (for lateral I3 and I4 incisors) through final maturation stages, as also evidenced in analyzed 1 year-old incisors. No significant changes were found to occur in the levels of carbonate substitution in dentin, with the exception of a small increase in mantle dentin at later maturation stages. The relative type of carbonate substitution (substitution for phosphate or hydroxyl ions or presence of labile carbonate) was also not found to vary significantly during maturation, again with the exception of a decrease in the relative A type carbonate (substitution for hydroxyl ions) in mantle dentin at later stages. When compared to the existing data on bone maturation, these results show a higher and steeper mineral:matrix increase, but lower crystallinity increase and no changes in carbonate substitution, whereas in bone the carbonate content has been described to increase or decrease, depending on the model used. While the same tissue age-dependent variations in mineral properties were observed on the left and right incisors as groups of samples, there was not a 1:1 correspondence in the distribution of these properties values between individual left incisors and their right counterparts. This suggested that factors such as reproducibility in the sectioning plane orientation may contribute to similar studies’ outcomes. Additionally, the presence of functional interfaces around the mineralization front and the dentinoenamel junction was shown by the distribution of mineral:matrix and crystallinity at the respective sites for all maturational stages.
Based on the FTIR imaging results approximately 80μm wide dentin areas of mantle dentin and of circumpulpal dentin immediately adjacent to mantle at successive maturational stages were identified on fetal bovine incisors and specimens from these areas were microdissected. Matrix properties (Chapter III), matrix relative content in phosphoproteins and overall phosphorylation (Chapter IV) were subsequently analyzed in these dentin specimens. The relative water content of the matrix was found to be greatly decreased during dentin maturation, again mainly during the initial maturational stages, by roughly the same amount for mantle and for circumpulpal dentin. A significant shift of the peak of the matrix main vibrational mode (Amide I) from early to later maturational stages that was identified suggested a change in the matrix secondary structure that is probably associated with the changes in the matrix hydration. The relative total matrix phosphate content of similar microdissected specimens presented a significant increase, while the amino acid analysis of the same specimens did not support any significant difference in phosphoprotein content of the matrix during dentin maturation. These results gave evidence for a continued phosphorylation of phosphoproteins in mineralizing dentin, which implied an involvement of phosphoproteins in later maturational stages.
When the same FTIRI analysis was applied to developing dentin of 6 day-old mouse molars (Chapter V), a continuous change in the mineral:matrix was shown occuring in a range much lower than in fetal bovine incisors. Crystallinity did not show a steady increase as in the fetal bovine incisors. Reproducibility in the mineral
changes results was found to be highly dependent on the specimen orientation during sectioning.
These studies provided a refined model for analysis and a description of mineral and matrix changes separately for mantle and circumpulpal dentin at true successive maturational stages and without any superimposed histological variability. It also provided a baseline for studying changes in developing dentin in other species (human, rodents) and in the presence of disease, where the relevant studies would provide a better understanding of pathogenesis in dental tissues. The present data will equally complement data on mineralization in reparative dentin, with a view to maximizing the reparative dentin mineral and biomechanical properties in future studies. The described matrix properties findings supported a pronounced decrease in the dentin water content that had not been clearly described for mantle and circumpulpal dentin, which can be quantitatively estimated by weight determination in future studies. These studies also provided evidence of a continued matrix phosphorylation in mineralizing dentin, a fact that merits further investigation by analysis of casein kinase activity in dentin or of post-translational modifications of matrix proteins from different tissue age dentin specimens. The possibilities and limitations of general methodology based on FTIRI analysis application on developing mouse dentin were also investigated for the first time. If that methodology is optimized, probably by using higher resolution analytic techniques and a highly reproducible specimen preparation method, for the purpose, the transfer of all the mentioned kinds of studies to animals genetically targeted for one or more matrix proteins will be possible.
ACKNOWLEDGMENTS
The author is thankful to the advisors (A.L. Boskey and J. Timothy Wright) and the other members of the thesis committee (P. Flood, R. Arnold, R. Murray, W. Duarte) for their guidance and support. He is also very thankful to L. Lukashova for the great effort she contributed to this thesis. Without their support this thesis would not have been realized. The studies described were supported by NIH