Abstract
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.
Highlights
Drug repositioning is a research method that searches for new drug effects using existing drugs for which the safety and pharmacokinetics in humans have already been confirmed, increasing the practical applications
Since Alkaline Phosphatase (ALP) is used as the initial marker for the differentiation of mesenchymal cells into hard tissue-forming cells such as osteoblasts [6], we investigated the effects of MDZ and bone morphogenetic protein-2 (BMP-2) on ALP activity in the C2C12 cells
With 500 ng/mL recombinant human bone morphogenetic protein-2 (rhBMP-2), used in our previous study [8], we initially explored the optimal concentration of MDZ in a range from 0.1 to 100 μM and found that 10 μM MDZ has the highest ALP activity in C2C12 cells (Figure S1)
Summary
Drug repositioning is a research method that searches for new drug effects using existing drugs for which the safety and pharmacokinetics in humans have already been confirmed, increasing the practical applications. The greatest advantages of drug repositioning are certainty about the safety and pharmacokinetics at the clinical level and low cost, since existing data can be used. We were motivated to use mesenchymal stem cells (MSCs) to discover the potential for drug repositioning for future bone treatments and organ regenerative medicine. In research on osteogenic differentiation using MSCs, differentiation from MSCs to osteoblasts is regulated by many molecular factors and mechanical stimuli. Human mesenchymal stem cells (hMSCs) differentiate into osteoprogenitor cells, preosteoblasts, osteoblasts, and osteocytes under appropriate culture conditions, and express several important osteogenic genes during this differentiation process [2]. In vitro differentiation of MSCs depends on culture conditions, and growth factors, such as those of the transforming growth factor β (TGF-β) family, significantly affect MSC differentiation [3,4]
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