Self-assembled monolayer facilitates epithelial-mesenchymal interactions mimicking odontogenesis.

Cell-cell interactions are vital for embryonic organ development and normal function of differentiated cells and tissues. In this study we have developed a self-assembled monolayer-based co-culture system to study tooth morphogenesis. Specifically, we designed a 2-D microenvironment present in the dental tissue by creating a well-structured, laterally organized epithelial and mesenchymal cell co-culture system by patterning the cell-attachment substrate. Chemical modifications were used to develop tunable surface patterns to facilitate epithelial-mesenchymal interactions mimicking the developing tooth. Such a design promoted interactions between monolayer's of the 2 cell types and provided signaling cues that resulted in cellular differentiation and mineralized matrix formation. Gene expression analysis showed that these co-cultures mimicked in-vivo conditions than monolayer cultures of a single cell type.

Dentin matrix protein 4, a novel secretory calcium-binding protein that modulates odontoblast differentiation.

Formation of calcified tissues is a well regulated process. In dentin, the odontoblasts synthesize several biomolecules that function as nucleators or inhibitors of mineralization. To identify genes that are odontoblast-specific, a subtractive hybridization technique was employed that resulted in the identification of a previously undescribed novel gene synthesized by the odontoblasts. Based on the nomenclature in our laboratory, this gene has been named dentin matrix protein 4 (DMP4). The protein encoded by mouse DMP4 cDNA contained 579 amino acids, including a 26-amino acid signal peptide. Analysis of the protein sequence demonstrated the presence of a Greek key calcium-binding domain and one conserved domain of unknown function in all the species examined thus far. Calcium binding property was confirmed by (45)Ca binding assays and the corresponding change in conformation by far-ultraviolet circular dichroism. Northern analysis demonstrated high expression levels of a single 3-kb mRNA transcript in tooth, whereas low expression levels were detected in other tissues. In situ hybridization analysis showed high expression levels of DMP4 in odontoblasts and low levels in osteoblasts and ameloblasts during tooth development. Gain and loss of function experiments demonstrated that DMP4 had the potential to differentiate mesenchymal precursor cells into functional odontoblast-like cells.

Identification of differentially expressed cDNA transcripts from a rat odontoblast cell line.

Odontoblasts and osteoblasts are two among the myriads of cell types present in the craniofacial complex. Both have a common ectomesenchymal origin and secrete macromolecules that are necessary for the formation of dentin and alveolar bone via matrix-mediated mechanisms. The mineralized matrices of bone and dentin differ in morphology and function but several mineral associated proteins, formerly thought to be tissue specific, have been found to be common in both tissues. To decipher the complex molecular mechanisms involved in mineralized dentin formation, the suppressive subtraction hybridization (SSH) approach has been used to identify the genes expressed by polarized odontoblasts. Employing SSH, 187 cDNA clones were identified from the subtracted cDNA library. Many of these genes have not been previously reported to be expressed by terminally differentiated odontoblasts. Genes were classified into seven groups based on the predicted function of the encoded proteins: extracellular matrix; cytoskeletal components, molecules involved in adhesion and cell-cell interaction; metabolic enzymes, transporters, ion channels; protein processing, protein transport and protein folding molecules; nuclear proteins (transcription factors, DNA processing enzymes); signaling molecules and genes of yet unknown function. Northern blot and in situ hybridization analysis performed for five putative novel genes and one new isoform of amelogenin revealed differential expression levels in the osteoblasts, ameloblasts and the odontoblasts of the developing rat molars. Some of the known genes isolated from this enriched pool were the cleavage products of dentin sialophosphoprotein (DSPP) namely, phosphophoryn (PP) and dentin sialoprotein (DSP). Interestingly amelogenin, ameloblastin and enamelin were also expressed in the odontoblasts during dentin formation.