METHODOLOGICAL APPROACH TO IN VITRO DIFFERENTIATION OF HUMAN ODONTOBLAST-LIKE CELLS

Objectives: In vitro differentiation model system to odontoblast-like cells can provide a homogeneous cell population under conditions that limit the number of variables the cell environment, and thus simplify the analysis of the complex processes that constitute the cell response. The system also provide excellent opportunities, in order to have a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation, cell behavior in dental materials as well as the molecular events involved in the intracellular signaling pathways and dentin bio mineralization.
To establish an in vitro cell model that preserves the most of physiological, biochemical and genetic characteristics of human odontoblast-like cells.
Methods: Human Dental Pulp cells (hDPCs) were obtained from healthy human third molars. After analyzing by flow cytometry the expression of dental mesenchymal cells markers, we proceeded to induce differentiation with TGF-b1 supplemented medium. We evaluated the expression dentin sialophosphoprotein (DSSP) and dentin matrix protein-1 (DMP-1) odontoblast markers by immunocytochemistry. In addition, we determined alkaline phosphatase (ALP) activity and nodule formation by alizarin red S (ALZ) and Von Kossa staining. Masson's trichrome stain was used to assess the deposition of extracellular matrix.
Results: Flow cytometry analysis revealed that hDPCs were CD73+, CD90+, CD105+, CD14-, CD20-, CD34-, CD45-, a set of mesenchymal cell markers, confirming its origin. Odontoblast-like cell showed a positive immunostaining for DSSP and DMP-1. The odontogenic stimulus induced an increase in the formation of calcified nodules and extracellular matrix deposition. ALP activity was also markedly stimulated by differentiation culture medium, which reached a maximum value at 8 days.
Conclusions: This study evidenced the isolation of mesenchymal progenitors cells with potential enough to promote odontoblast-like cells differentiation in vitro cultures. These results are useful for further developments involving tissue engineering and biocompatibility studies of dental materials.