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

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.

Bioinspired Anisotropic Chitosan Hybrid Hydrogel

“…In addition to the outstanding mechanical properties, the ACHH also demonstrates excellent biocompatibility. As an immortalized mouse skeletal muscle cell line, C2C12 as model cells have been widely used for undifferentiated mesenchymal cells to study the differentiation of myoblasts and osteoblasts . To evaluate the cytocompatibility of ACHH, C2C12 were cultured in the medium containing ACHH.…”

“…As an immortalized mouse skeletal muscle cell line, C2C12 as model cells have been widely used for undifferentiated mesenchymal cells to study the differentiation of myoblasts and osteoblasts. 57 To evaluate the cytocompatibility of ACHH, C2C12 were cultured in the medium containing ACHH. The results of cell viability measurement are shown in Figure 5a.…”

KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK

“…The treatment of C2C12 myoblasts with BMP2, playing a crucial role in skeletal development and limb formation [ 34 ], is a recognized model to induce cell differentiation into an OB lineage [ 16 , 17 ]. Indeed, while untreated C2C12 cells formed myotubes and expressed the myogenic marker desmin [ 35 ] after 6 days in a low serum medium, the treatment with BMP2 (300 ng/mL) reduced the presence of desmin-positive cells and converted the cell morphology into an OB-like round shape ( Figure 4 A).…”

“…Interestingly, the three herbal extracts combined in the KYMASIN UP and their active compounds have been reported to modulate OBs and/or OCs activities as well as bone structure [ 9 , 10 , 11 , 12 , 13 ]. The aim of the present study is to assess the effectiveness of KYMASIN UP in the differentiation process and activity of OCs and OBs by using established in vitro models consisting of the RAW 264.7 monocyte cell line conditioned with RANKL (receptor activator of nuclear factor kappa-Β ligand) protein [ 14 , 15 ] and C2C12 myoblasts treated with BMP2 (bone morphogenetic protein 2) [ 16 , 17 ], respectively. The molecular mechanisms underlying the effects of KYMASIN UP and its efficacy in human primary OBs were also investigated.…”

Development and Characterization of Alkaline Phosphatase-Positive Human Umbilical Cord Perivascular Cells

“…In in vitro studies, hard tissue-forming cells such as C2C12, MC3T3-E1, and PPU7 have generally been observed to form mineralized nodules when they are cultured in a mineralization medium [ 24 , 45 , 46 ]. When HUCPVCs were cultured in conditioned media derived from cultures of human-bone-marrow derived mesenchymal stromal cells, mineralized nodules, suggestive of osteogenic differentiation, formed in HUCPVCs [ 19 ].…”