Research Paper
Bone Regeneration
The effect of nicotine on osteoinduction by recombinant human bone morphogenetic protein 2

https://doi.org/10.1016/j.ijom.2014.02.014Get rights and content

Abstract

Nicotine, one of the constituents of tobacco, is known to have an adverse effect on human health. We sought to clarify the interaction between nicotine and recombinant human bone morphogenetic protein 2 (rhBMP-2) in terms of osteogenesis in vitro and osteoinduction in vivo. Nicotine did not inhibit or stimulate alkaline phosphatase (ALP) activity or the amount of osteocalcin in C2C12 cells in the presence of rhBMP-2 in vitro. Ectopic bone formation using a collagen sponge containing rhBMP-2 was evaluated with and without nicotine after 21 days using radiographic, histological, biochemical, and immunohistochemical analyses. ALP activity in the medium-dose group (2.2 ± 0.9 IU/mg protein; P = 0.047) and the high-dose group (2.0 ± 0.1 IU/mg protein; P = 0.03) was significantly lower than in the control group. The calcium content in the medium-dose group (35.4 ± 12.9 μg/mg tissue; P = 0.0099) and high-dose group (34.8 ± 10.5 μg/mg tissue; P = 0.006) was significantly lower than in the control group. The number of vascular endothelial growth factor-positive cells in the high-dose group (671.9 ± 57.3 cells/mm2; P = 0.03) was significantly lower than in the control group. Results showed that nicotine did not inhibit the stimulatory effect of rhBMP-2 in vitro, but a high dose of nicotine inhibited bone formation in vivo by adversely affecting vascularization.

Section snippets

Cytotoxicity evaluation assay (MTS test)

Cytotoxicity was evaluated using the Cell Titer 96 One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). The reaction is based on the bioreduction of the substrate 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) into a brown formazan product by mitochondrial dehydrogenases of viable cells.29 C2C12 cells (American Type Culture Collection) were seeded at 5 × 102 cells per well in a 96-well plate containing Dulbecco's modified Eagle's

Cytotoxicity of nicotine

The addition of nicotine did not inhibit cellular proliferation at any concentration. There was no difference among groups, including the control (Fig. 1A).

ALP and osteocalcin activity

There was also no significant difference in ALP activity or the amount of osteocalcin among groups (Fig. 1B and C). These results indicate that nicotine had no effect on the osteogenic differentiation of C2C12 cells by rhBMP-2.

von Kossa staining

To evaluate the osteogenic differentiation of C2C12 cells by rhBMP-2, the calcium deposits were stained by the von

Discussion

The results of the in vitro experiments showed that nicotine at a concentration range of 0.01–10 μM did not have a pronounced effect on the proliferation of C2C12 cells based on the MTS assay, a type of metabolism assay. However, it has recently been reported that metabolic assays may not accurately reflect proliferation due to non-linear and miscorrelating changes in cell numbers and activity over time in culture.31 Therefore, the results should be interpreted in light of this limitation of the

Funding

This study was supported by grants from the Smoking Research Foundation in Japan.

Competing interests

None declared.

Ethical approval

Not required.

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