Potential for Drug Repositioning of Midazolam for Dentin Regeneration.

Drug repositioning promises the advantages of reducing costs and expediting approvalschedules. An induction of the anesthetic and sedative drug; midazolam (MDZ), regulatesinhibitory neurotransmitters in the vertebrate nervous system. In this study we show the potentialfor 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 bonemorphogenetic protein 2, and the combination of MDZ with transforming growth factor-beta 1. Thedifferentiation 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 engineeringlevels. The MDZ-only treatment enhanced the alkaline phosphatase activity and mRNA levels ofodontoblast differentiation marker genes, and precipitated nodule formation containing a dentinspecificprotein (dentin phosphoprotein). The nodules consisted of randomly orientedhydroxyapatite nanorods and nanoparticles. The morphology, orientation, and chemicalcomposition of the hydroxyapatite crystals were similar to those of hydroxyapatite that hadtransformed from amorphous calcium phosphate nanoparticles, as well as the hydroxyapatite inhuman molar dentin. Our investigation showed that a combination of MDZ and PPU-7 cellspossesses high potential of drug repositioning for dentin regeneration.

Repurposing MDZ as a tool for tissue regeneration in dental cells.

BACKGROUND: Several recent studies have focused on the utility of drug repurposing to expand clinical application of approved therapeutics. Here, we investigate the efficacy of midazolam (MDZ) and cytokines for regenerating calcified tissue, using immortalized porcine dental pulp (PPU7) and mouse skeletal muscle derived myoblast (C2C12) cells, with the goal of repurposing MDZ as a new treatment to facilitate calcified tissue regeneration. HIGHLIGHTS: We noted that PPU7 and C2C12 cells cultured with various MDZ regimens displayed increased bone morphogenic protein (BMP-2), transforming growth factor beta (TGF-ß), and alkaline phosphatase activity. These increases were highest in PPU7 cells cultured with MDZ alone, and in C2C12 cells cultured with MDZ and BMP-2. PPU7 cells cultured under these conditions demonstrated markedly elevated expression of odontoblastic gene markers, indicating their likely differentiation into odontoblasts. Expression levels of osteoblastic gene markers also increased in C2C12 cells, suggesting that MDZ potentiates the effect of BMP-2, inducing osteoblast differentiation in these cells. Newly formed calcified deposits in both PPU7 and C2C12 cells were identified as hydroxyapatite via crystallographic and crystal engineering analyses. CONCLUSION: MDZ increases ALP activity, inducing expression of specific marker genes for both odontoblasts and osteoblasts while promoting hydroxyapatite production in both PPU7 and C2C12 cells. These responses were cell type specific. MDZ treatment alone could induce these changes in PPU7 cells, but C2C12 cell differentiation required BMP-2 addition.

Combined Effect of Midazolam and Bone Morphogenetic Protein-2 for Differentiation Induction from C2C12 Myoblast Cells to Osteoblasts.

In drug repositioning research, a new concept in drug discovery and new therapeutic opportunities have been identified for existing drugs. Midazolam (MDZ) is an anesthetic inducer used for general anesthesia. Here, we demonstrate the combined effects of bone morphogenetic protein-2 (BMP-2) and MDZ on osteogenic differentiation. An immortalized mouse myoblast cell line (C2C12 cell) was cultured in the combination of BMP-2 and MDZ (BMP-2+MDZ). The differentiation and signal transduction of C2C12 cells into osteoblasts were investigated at biological, immunohistochemical, and genetic cell levels. Mineralized nodules formed in C2C12 cells were characterized at the crystal engineering level. BMP-2+MDZ treatment decreased the myotube cell formation of C2C12 cells, and enhanced alkaline phosphatase activity and expression levels of osteoblastic differentiation marker genes. The precipitated nodules consisted of randomly oriented hydroxyapatite nanorods and nanoparticles. BMP-2+MDZ treatment reduced the immunostaining for both α1 and γ2 subunits antigens on the gamma-aminobutyric acid type A (GABAA) receptor in C2C12 cells, but enhanced that for BMP signal transducers. Our investigation showed that BMP-2+MDZ has a strong ability to induce the differentiation of C2C12 cells into osteoblasts and has the potential for drug repositioning in bone regeneration.