J Dent Anesth Pain Med.  2018 Feb;18(1):9-17. 10.17245/jdapm.2018.18.1.9.

Effect of remifentanil on pre-osteoclast cell differentiation in vitro

• Jeon HO
• Choi IS
• Yoon JY
• Kim EJ
• Yoon JU
• Cho AR
• Kim HJ
• Kim CH

• KMID: 2405378
• DOI: http://doi.org/10.17245/jdapm.2018.18.1.9

Abstract

BACKGROUND
The structure and function of bone tissue is maintained through a constant remodeling process, which is maintained by the balance between osteoblasts and osteoclasts. The failure of bone remodeling can lead to pathological conditions of bone structure and function. Remifentanil is currently used as a narcotic analgesic agent in general anesthesia and sedation. However, the effect of remifentanil on osteoclasts has not been studied. Therefore, we investigated the effect of remifentanil on pre-osteoclast (pre-OCs) differentiation and the mechanism of osteoclast differentiation in the absence of specific stimulus.
METHODS
Pre-OCs were obtained by culturing bone marrow-derived macrophages (BMMs) in osteoclastogenic medium for 2 days and then treated with various concentration of remifentanil. The mRNA expression of NFATc1 and c-fos was examined by using real-time PCR. We also examined the effect of remifentanil on the osteoclast-specific genes TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. Finally, we examined the influence of remifentanil on the migration of pre-OCs by using the Boyden chamber assay.
RESULTS
Remifentanil increased pre-OC differentiation and osteoclast size, but did not affect the mRNA expression of NFATc1 and c-fos or significantly affect the expression of TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. However, remifentanil increased the migration of pre-OCs.
CONCLUSIONS
This study suggested that remifentanil promotes the differentiation of pre-OCs and induces maturation, such as increasing osteoclast size. In addition, the increase in osteoclast size was mediated by the enhancement of pre-OC migration and cell fusion.

Keyword

Cell Fusion; Cell Migration; Osteoclasts; Remifentanil

MeSH Terms

Anesthesia, General
Bone and Bones
Bone Remodeling
Cathepsin K
Cell Differentiation*
Cell Fusion
Cell Movement
In Vitro Techniques*
Macrophages
Osteoblasts
Osteoclasts
Real-Time Polymerase Chain Reaction
Receptors, Calcitonin
RNA, Messenger
Cathepsin K
RNA, Messenger
Receptors, Calcitonin

Figure

• 

  Fig. 1 Remifentanil does not affect the cytotoxicity or cell proliferation of pre-osteoclasts (pre-OCs). (A) Pre-OCs were incubated in medium containing the indicated concentrations of remifentanil (0–100 ng/ml) for 24 h. Cell viability was evaluated by using the MTT assay, (B) Pre-OCs were cultured in osteoclastogenic medium (30 ng/ml M-CSF + 100 ng/ml RANKL) for 3 days in the absence or presence of remifentanil (up to 100 ng/ml). Cell proliferation was measured by using the MTT assay.

• 

  Fig. 2 Remifentanil increased pre-osteoclast (pre-OC) differentiation and the size of osteoclasts. (A) Analysis of osteoclast differentiation by TRAP staining. Pre-OCs treated with M-CSF alone or M-CSF and RANKL were cultured with remifentanil (0, 10, and 50 ng/ml) for 2 and 3 days and stained for TRAP activity, (B) The size of osteoclasts was measured. All quantitative data are the mean ± SD of three independent experiments. (**P < 0.01).

• 

  Fig. 3 The mRNA expression of NFATc1 and c-fos was not affected by remifentanil during osteoclast differentiation. (A–B) The mRNA expression of NFATc1 and c-fos was analyzed by using quantitative real-time PCR. Pre-osteoclasts were cultured with the indicated doses of remifentanil in M-CSF alone or M-CSF and RANKL for 2 and 4 days.

• 

  Fig. 4 Remifentanil did not affect significantly gene expression related to osteoclast differentiation in pre-osteoclasts. (A–C) The mRNA expressions of TRAP, cathepsin K (CTK), and calcitonin receptor (CTR) were determined by using quantitative real-time PCR. TRAP, CTR, and CTK are osteoclastic differentiation markers, (D) The mRNA expression of DC-STAMP, known as a master regulator of osteoclast cell fusion, was analyzed by using quantitative real-time PCR.

• 

  Fig. 5 The migration of pre-osteoclasts (pre-OCs) was evaluated by using a Boyden chamber assay. (A) Pre-OCs in DMEM were added to the upper chamber and remifentanil (10 and 50 ng/ml) in DMEM was added into the lower chamber. The pre-OCs that migrated to the lower chamber were fixed and stained with hematoxylin and eosin solution, (B) The number of migrated cell numbers were counted. All quantitative data are the mean ± SD of three independent experiments (***, P < 0.001).

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