Abstract
Drug repositioning promises the advantages of reducing costs and expediting approval schedules. An induction of the anesthetic and sedative drug; midazolam (MDZ), regulates inhibitory neurotransmitters in the vertebrate nervous system. In this study we show the potential for drug repositioning of MDZ for dentin regeneration. A porcine dental pulp-derived cell line (PPU-7) that we established was cultured in MDZ-only, the combination of MDZ with bone morphogenetic protein 2, and the combination of MDZ with transforming growth factor-beta 1. The differentiation of PPU-7 into odontoblasts was investigated at the cell biological and genetic level. Mineralized nodules formed in PPU-7 were characterized at the protein and crystal engineering levels. The MDZ-only treatment enhanced the alkaline phosphatase activity and mRNA levels of odontoblast differentiation marker genes, and precipitated nodule formation containing a dentin-specific protein (dentin phosphoprotein). The nodules consisted of randomly oriented hydroxyapatite nanorods and nanoparticles. The morphology, orientation, and chemical composition of the hydroxyapatite crystals were similar to those of hydroxyapatite that had transformed from amorphous calcium phosphate nanoparticles, as well as the hydroxyapatite in human molar dentin. Our investigation showed that a combination of MDZ and PPU-7 cells possesses high potential of drug repositioning for dentin regeneration.
Highlights
Because alkaline phosphatase (ALP) is considered the initial marker for the differentiation of mesenchymal cells into hard tissue-forming cells such as osteoblasts or odontoblasts [24,25], we investigated the effects of MDZ on ALP activity in the PPU-7 cell line by using a quantitative colorimetric method with a p-nitrophenylphosphate as the substrate and examined the combination of MDZ (0-10 μM) with a recombinant human BMP2 (500 ng/mL) or a recombinant human TGF-β1 (1 ng/mL) (Figure 1b)
When the ALP activity level of the control (i.e., 0 μM MDZ, 0 ng/mL recombinant human BMP2 (rhBMP2), 0 ng/mL rhTGF-β1) was set at 1.0, the addition of MDZ-only significantly enhanced ALP activity in PPU-7 cells in a concentration-dependent manner, especially the ALP activity at 10 μM MDZ (i.e., 10 μM MDZ, 0 ng/mL rhBMP2, 0 ng/mL rhTGF-β1), which was 1.75-fold higher than the control
ALP staining for the mineral-induced PPU-7 cells displayed blue colored staining images (Figure 1c)
Summary
Applications of tissue engineering are founded on three components; a cell source, a scaffold, and bioactive molecules. In the field of dental tissue engineering, various soft and hard dental tissues have been regenerated in vitro using stem cells [1]. Dental pulp stem cells (DPSCs) have been isolated with various techniques and used for studies related to the cell differentiation potential and scaffolding for tissue regeneration [2,3]. DPSCs can differentiate into multiple cell types, including odontogenic and osteogenic cells, osteocytes, chondrocytes, vascular cells, neurons, and hepatocytes. DPSCs are used to generate induced pluripotent stem (iPS) cells
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