The analysis of ameloblastin and a human tooth germ cell culture system

The protein ameloblastin is the second most common tooth enamel protein, yet there is little data on this protein or gene sequence. The bovine ameloblastin gene sequence was determined by library screening and IPCR amplification of alternatively spliced mRNAs. This sequence information resulted in the identification of a new splice variant with unique predicted phosphorylation sites that were either spliced in or spliced out. Functional analysis studies were initiated using immunoperturbation of a mouse tooth germ organ culture and an antibody against the protein ameloblastin. These immunoperturbation experiments of the cultured tooth germs established a possible function of ameloblastin as a negative suppressor of enamel formation. In order to further understand the ameloblastin protein, a novel “ameloblast-like” primary cell culture was established and characterized. The cell culture system was used to identify two different cell types, cobble stone shaped epithelial cells (CABS) and spindle shaped cells (SPABS). The cells were characterized by identifying enamel specific mRNA expression and protein secretion using PCR, immunohistochemistry and Western Blot. Both cell types showed similar “ameloblast-like” characteristics in relation to protein and mRNA expression. Immunohistochemical analysis of amelogenin mRNA expression, showed a nuclear staining pattern in the CAB cells with an anti-amelogenin antibody, where the SPABS showed cytoplasmic staining. Another difference was in the reliance of CAB cells on calcium, which was similar to that found in keratinocytes in culture. The localization pattern and functional significance of these results will be analyzed in future studies. These studies will also include that analyzing the predicted phosphorylation pattern of ameloblastin through cell culture. Although further studies are necessary to confirm the possible function of ameloblastin, and it's role in ameloblast function, the characterization of ameloblastin splice variants, and the development of a primary cell ameloblast cell culture system, descried in this dissertation, are a major advance in our understanding of the biology of tooth enamel formation. The results of this dissertation project lead to further advances on the ameloblastin protein, and show major advances in the field of tooth development with the establishment of a novel cell culture system.