Effect of inducible bone morphogenetic protein 2 expression on the osteogenic differentiation of dental pulp stem cells in vitro.

Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily, it is known to be a factor involved in skeletal development and capable of inducing in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) isolated from extracted third molar teeth are an ideal resource for bone tissue engineering and regeneration applications, due to their convenient isolation, safe cryopreservation, and easy maintenance in cell cultures. The aims of this study were to deliver BMP-2 under control of the tetracycline-inducible (tet-on) promoter into dental pulp stem cells and to examine whether these BMP-2 expressing cell lines are capable of promoting osteogenic differentiation in vitro. BMP-2 gene was cloned into the lentiviral transfer plasmid pTet-IRES-EGFP and used to establish the DPSC-BMP-2 cell line. DPSC, DPSC-GFP (mock) and DPSC-BMP-2 cell lines were cultured in growth medium or osteogenic medium in the presence or absence of 100 ng/ml doxycycline. To assess differentiation, alkaline phosphatase activity, calcium accumulation and gene transcription levels of different genes involved in osteogenic differentiation (BMP-2, Runx2, alkaline phosphatase, and noggin) were measured. Doxycycline-induced BMP-2 expression induced the differentiation of DPSCs into the preosteoblastic stage but could not favor the further maturation into osteoblasts and osteocytes. We found that while Runx2 gene transcription was continuously upregulated in doxycycline-treated DPSC-BMP-2 cells, the alkaline phosphatase activity and the accumulation of minerals were reduced. As a result of the increased BMP-2 expression, the transcription level of the BMP antagonist noggin was also upregulated, and probably caused the observed effects regarding alkaline phosphatase (ALP) activity and mineral deposition. Our study shows that this system is effective in controlling transgene expression in DPSC cell line. Exploration of all known factors affecting osteogenic differentiation and their interactions is of major importance for the field of regenerative medicine. As the metabolic reaction to the upregulated transgene transcription appears to be cell line-specific, a wrongly selected target gene and/or regulation system could have adverse effects on differentiation.

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Declaration of competing interest The authors declare that there is no conflict of interest.