Nephronectin Stimulates the Differentiation of MDPC-23 Cells into an Odontoblast-like Phenotype

Introduction: The present study investigated the in vitro effects of nephronectin (Npnt) on the proliferation, differentiation, and mineralization of a rat odontoblast-like cell line (MDPC-23 cells). Methods: MDPC-23 cells were cultured on Npnt-coated polystyrene or in the presence of soluble Npnt. Cell proliferation was analyzed using a Cell Counting Kit-8 kit (Dojindo, Kumamoto, Japan). Alkaline phosphatase (ALP) activity was quantified using an ALP activity assay. A reverse transcription polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression level of odontogenic markers and integrin(s). Alizarin red staining was conducted to quantify the calcium deposition. Results: Soluble Npnt had no adverse effect on the proliferation of MDPC-23 cells, but it exhibited concentration-dependent inhibitory activity toward differentiation. In contrast, coated Npnt promoted cell proliferation dramatically and significantly up-regulated the mRNA expression of odontogenesis-related genes; moreover, mRNA expression of integrin alpha 1, alpha 3, alpha 5, beta 1 and beta 5 was found to be augmented. MDPC-23 cells cultured on Npnt-coated polystyrene displayed markedly higher ALP activity as early as day 3 after inoculation. In addition, mineralization was accelerated on Npntcoated polystyrene. Conclusions: Npnt in its immobilized form enhanced the proliferation of MDPC-23 cells and induced this odontoblastic precursor cell line to differentiate into a mineralizing phenotype.