J Dent Anesth Pain Med.  2019 Apr;19(2):91-99. 10.17245/jdapm.2019.19.2.91.

Remifentanil promotes osteoblastogenesis by upregulating Runx2/osterix expression in preosteoblastic C2C12 cells

• Yoon JY
• Kim TS
• Ahn JH
• Yoon JU
• Kim HJ
• Kim EJ

• KMID: 2444753
• DOI: http://doi.org/10.17245/jdapm.2019.19.2.91

Abstract

BACKGROUND
The imbalance between osteoblasts and osteoclasts can lead to pathological conditions such as osteoporosis. It has been reported that opioid adversely affect the skeletal system, but it is inconsistent. Remifentanil is currently used as an adjuvant analgesic drug in general anesthesia and sedation. The aim of the present study was to investigate the effect of remifentanil on the osteoblast differentiation and mechanism involved in this effect.
METHODS
The C2C12 cells (mouse pluripotent mesenchymal cell line) were used as preosteoblast. Osteoblastic differentiation potency was determined by alkaline phosphatase (ALP) staining. C2C12 cell migration by remifentanil was evaluated using Boyden chamber migration assay. The expression of Runx2 and osterix was evaluated by RT-PCT and western blot analysis to investigate the mechanism involved in remifentanil-mediated osteoblast differentiation.
RESULTS
ALP staining showed that remifentanil increased significantly osteoblast differentiation. In Boyden chamber migration assay, C2C12 cell migration was increased by remifentanil. RT-PCR and western blot analysis showed that the expression of Runx2 and osterix was upregulated by remifentanil.
CONCLUSIONS
We demonstrated that remifentanil increased osteoblast differentiation in vitro by upregulation of Runx2 and osterix expression. Therefore, remifentanil has the potential for assisting with bone formation and bone healing.

Keyword

Osteoblasts; Osterix; Remifentanil; Runx2

MeSH Terms

Alkaline Phosphatase
Anesthesia, General
Blotting, Western
Cell Movement
In Vitro Techniques
Osteoblasts
Osteoclasts
Osteogenesis
Osteoporosis
Up-Regulation
Alkaline Phosphatase

Figure

• 

  Fig. 1 Effect of remifentanil on cytotoxicity and cell proliferation in C2C12 cells. (A) C2C12 cells were incubated in medium containing indicated concentrations of remifentanil (0–100 ng/ml) for 24 h. Cell viability was evaluated by MTT assay. (B) C2C12 cells were cultured in DMEM and 100 µg of BMP-2 for 3 days in the presence of indicated doses of remifentanil (0–100 ng/ml). Cell proliferation was measured at daily intervals by MTT assay. CON: control

• 

  Fig. 2 Effect of remifentanil on osteoblast differentiation in preosteoblastic C2C12 cells. (A) C2C12 cells were cultured in osteogenic media (DMEM and 100 µg/ml BMP-2) for the indicated number of days. The cells were treated with remifentanil (0, 1 and 10 ng/ml). At day 1 and day 3, osteoblast differentiation was examined by alkaline phosphatase (ALP) staining. (B) ALP-positive area of C2C12 cells was quantified by densitometry of triplicate experiments. All quantitative data are means ± SD of three independent experiments (*, P < 0.05). CON: control; BMP-2: bone morphogenetic protein-2

• 

  Fig. 3 Cell migration of C2C12 cell was evaluated using Boyden chamber migration assay. (A) The C2C12 cells in DMEM media were added on the upper chamber and remifentanil (10 and 50 ng/ml) in DMEM media was added into the lower chamber. Migrated C2C12 cells in lower chamber were fixed and stained with crystal violet solution. (B) Migrated cell numbers were counted. All quantitative data are means ± SD of three independent experiments (**, P < 0.01, ***, P < 0.001). CON: control

• 

  Fig. 4 The expression of Runx2 and osterix was increased by remifentanil. (A) The mRNA expression of various osteoblastic differentiation markers such as Runx2, osteopontin (OPN), bone sialoprotein (BSP), collagen type I (COL1A1), osteocalcin and osterix were evaluated using RT-PCR. (B) Western blot analysis was performed to investigate the expression of Runx2 and osterix. (C, D) The protein level of Runx2 and osterix was quantified. All quantitative data are means ± SD of three independent experiments (*, P < 0.05). CON: control; BMP-2: bone morphogenetic protein-2

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