International Journal of Oral and Maxillofacial Surgery
Research PaperBone RegenerationThe effect of nicotine on osteoinduction by recombinant human bone morphogenetic protein 2
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.
References (40)
The role of nicotine in smoking-related cardiovascular disease
Prev Med
(1997)- et al.
Nicotine: a review of its role in atherosclerosis
J Am Coll Surg
(2001) - et al.
The effect of intra-arterial epinephrine and nicotine on gingival circulation
Oral Surg Oral Med Oral Pathol
(1981) - et al.
Bone induction by Escherichia coli-derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2
Br J Oral Maxillofac Surg
(2000) - et al.
Expression of bone morphogenetic protein-2 via adenoviral vector in C2C12 myoblasts induces differentiation into the osteoblast lineage
Biochem Biophys Res Commun
(1999) - et al.
Comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
(1999) - et al.
Combined delivery of BMP-2 and bFGF from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo
J Control Release
(2013) - et al.
Treatment of irradiated mandibles with mesenchymal stem cells transfected with bone morphogenetic protein 2/7
J Oral Maxillofac Surg
(2012) - et al.
Change in blood perfusion and bone healing induced by nicotine during distraction osteogenesis
Bone
(2008) - et al.
5-(3-Carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple water soluble formazans as cell-viability indicators
Bioorg Med Chem Lett
(1991)