Induction of Mesenchymal Stem Cell and Preosteoblast Differentiation: A Comparative Study Between Conditioned Medium and In Vitro Coculture.

Objective: To determine and compare the effects on differentiation, proliferation, and morphology of dental pulp-derived mesenchymal stem cells (DPSC) and preosteoblasts (pre-OBs) (Saos-2) that are either cocultured or treated with conditioned medium (CM) in vitro. Materials and Methods: In this experimental in vitro study, DPSC and Saos-2 cells were cocultured either directly-allowing physical contact-or indirectly, using a transwell system. These coculture systems were compared to treatment with CM from these cells over varying time periods. Morphological changes were evaluated using light microscopy, while cell proliferation was assessed with the resazurin colorimetric assay. Osteoblastic differentiation was analyzed by Alizarin Red staining and by quantifying changes in Runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OCN) gene expression via real-time RT-PCR. All experiments were performed in triplicate and data analyzed by Student's Results: Treatment with CM reduced the cell numbers for both cell types, whereas direct and indirect coculture led to an increase in cell numbers after 8 days of treatment. Morphologically, these treatments induced the formation of more elongated cells with greater cellular extensions compared to control groups. All experimental groups exhibited calcium nodule formation and increased relative expression of RUNX2, OSX, and OCN, consistent with induction of osteogenic differentiation. Conclusions: The results of this study suggest that both direct and indirect coculture of Saos-2 and DPSC cells, as well as treatment with CM, are effective in inducing morphological changes, proliferation, and osteoblastic differentiation. These strategies may therefore serve as useful tools for research into regenerative therapies aimed at repairing bone defects.