A Human Odontoblast Phenotype Isolated from the Dentin-Pulp Interface

Objectives: The understanding of dentin formation and other functions of odontoblasts was restricted so far, because in vitro cultivable odontoblasts are not available. It was the aim of this study to isolate a proliferating population of cells from the dentin-pulp interface of a human third molar expressing an odontoblast phenotype.
Methods: An intact third molar was obtained from a young patient (18 yrs), the pulp tissue was removed, and cells were scraped off the walls of the coronal pulp cavity. The explants were cultivated using routine culture techniques. Cell morphology or ultrastructure was studied by light microscopy (LM), scanning (SEM) or transmission electron microscopy (TEM). Protein expression was analyzed by routine Western blotting, and mineralization was observed after alizarin staining. Cell proliferation and the expression of surface markers were examined by flow cytometry. Differences between mean values were statistically analyzed by one-way ANOVA (p<0.05).
Results: Cell polarity typical for odontoblast morphology was identified by LM, SEM and TEM. Ultrastructural analysis of cultured cells indicated high synthetic and secretory activity including the extracellular deposition of fibrillar collagen. The base of the cell contains the nucleus, mitochondria, rough endoplasmic reticulum and the Golgi apparatus while secretory vesicles were localized to the cell process. The cells express the odontoblast-specific proteins dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1), and show mineralisation after alizarin red staining. Cell proliferation was observed over a long time period with an estimated doubling time of 48h. Finally, the cells express surface markers CD90, CD29, CD73, or CD105 characteristic of mesenchyme-derived cells.
Conclusions: This study presents the isolation and cultivation of a differentiated human odontoblast phenotype suitable for analyses of dentin formation, immune responses or sensory functions.