CHARACTERIZATION OF EXPANDED HUMAN ODONTOBLAST-LIKE CELLS

Objectives: Primary cultures from dental pulp help to understand the phenotypic features, cell differentiation process and matrix-mediated mineralization. Dental pulp cells can be differentiated in supplemented supportive medium that contain TGF-β1, among others reagents. The formation of extracellular matrix (ECM) of dentin is determined partly, by the expression of non-collagenous proteins as Small Integrin-Binding Ligand, N-linked Glycoprotein (SIBLING family), that includes osteopontin (OPN), bone sialoprotein, dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). These SIBLING proteins are believed to play key biological roles in dentin mineralization.
Evaluate the genotypic and phenotypic characteristics of odontoblast-like cells differentiated after being detached and re-seeded post-differentiation.
Methods: Dental pulp mesenchymal cells (DPMCs) from human third molars were isolated and differentiated for 21 days with culture medium supplemented with TGF-β1. The characteristics of differentiated cells were compared with those trypsinized and expanded using RT-PCR and immunohistochemistry. Expanded cells were named OLC-21 (Odontoblast-like cells differentiated for 21 days). Alizarin red, Von Kossa and alkaline phosphatase (ALP) assays were used to determine the cells mineralizing potential. Population doubling level (PDL) was calculated after hemocytometer cell counting.
Results: Primary differentiated and OCL-21 expanded cells expressed mRNA for ECM proteins like ALP, OPN, osteonectin, osteocalcin, DMP-1 and DSPP and also showed mineralization activity. The expression of DMP-1 and DSPP was detectable for immunohistochemistry. Cell proliferation analysis revealed significantly increased proliferative activity of OLC-21 compared with the primary cells before detaching.
Conclusions: The need to develop cell models of odontoblast differentiation and to identify markers specific for the odontoblast lineage is a useful tool for studying the mechanism involved in the terminal differentiation process of these post-mitotic cells. The results demonstrated that the trypsinization and detachment process of differentiated cells did not affect the proliferative and differentiation characteristics of this cell culture.