The goal of this project is to study the biological function of these dentin-specific phosphoproteins in the process of dentin biomineralization, thereby advancing our overall understanding of the process of odontoblast differentiation and dentinogenesis. This goal is based on an emerging body of literature showing that acidic proteins generally play a key role in direct regulation of crystal formation. Our hypothesis is that the dentin-specific phosphoproteins, DMP 1 and DPP(s), are markers for odontoblast differentiation which are important in the nucleation and regulation of hydroxyapatite formation during dentin extracellular matrix mineralization. Our first objective is to isolate and characterize the full-length cDNAs for DMP-1 and DPP(s). This will be accomplished by construction and screening of a mouse molar cDNA library. Once identified, the DMP-1 and DPP(s) cDNAs will be heterologously expressed in procaryotic and eukaryotic hosts, and the purified protein used to produce specific antibodies. These antibodies will be used to map the sequential expression patterns of DMP-1 and DPP(s) during odontoblast differentiation related to dentin extracellular matrix mineralization using immunohistochemistry. The expression of these dentin-specific phosphoproteins at a transcriptional level will also be examined using RT-PCR amplification and in situ hybridization techniques. Finally, the biological function of DMP-1 and DPP(s) will be investigated using antisense """"""""knockout"""""""" and antibody perturbation strategies monitoring the resulting mineral phase and matrix formation using electron microscopy, X-ray diffraction and electron microprobe analyses. These experiments will take advantage of a unique monolayer odontoblast cell culture system established in our laboratory to determine the functions of the dentin-specific phosphoproteins. This culture system has previously been shown to be conducive for the cytodifferentiation of odontoblasts from dental papilla mesenchyme cells, maintenance of the odontoblast phenotype, sequential expression and secretion of dentin extracellular matrix proteins, and dentin biomineralization. In addition, we have recently established immortalized mouse odontoblast and dental papilla mesenchyme cell lines at key stages of the odontoblast differentiation which will be used to determine the effect of modulation of dentin-specific phosphoprotein expression on DECM production and subsequent mineralization. These two key research tools will be used to investigate the biological function of dentin-specific phosphoproteins during odontoblast differentiation and dentin mineralization in this application.
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