The use of carbon nanotubes to induce osteogenic differentiation of human adipose-derived MSCs in vitro and ectopic bone formation in vivo

Carbon nanotubes (CNTs), one of the most concerned nanomaterials, with unique electrical, mechanical and surface properties, have been shown suitable for biomedical application. In this study, we evaluated attachment, proliferation, osteogenic gene expression, ALP/DNA, protein/DNA and mineralization of human adipose-derived stem cells cultured in vitro on multi-walled carbon nanotubes (MWNTs) and graphite (GP) compacts with the same dimension. Moreover, we assessed the effect of these two kinds of compacts on ectopic bone formation in vivo. First of all, higher ability of the MWNTs compacts to adsorb proteins, comparing with the GP compacts, was shown. During the conventional culture, it was shown that MWNTs could induce the expression of ALP, cbfa1 and COLIA1 genes while GP could not. Furthermore, alkaline phosphatase (ALP)/DNA and protein/DNA of the cell on the MWNTs compacts, was significantly higher than those of the cells on the GP compacts. With the adsorption of the proteins in culture medium with 50% fetal bovine serum (FBS) in advance, the increments of the ALP/DNA and protein/DNA for the MWNTs compacts were found respectively significantly more than the increments of those for the GP compacts, suggesting that the larger amount of protein adsorbed on the MWNTs was crucial. More results showed that ALP/DNA and protein/DNA of the cells on the two kinds of compacts pre-soaked in culture medium having additional rhBMP-2 were both higher than those of the cells on the samples re-soaked in culture medium with 50% FBS, and that those values for the MWNTs compacts increased much more. Larger mineral content was found on the MWNTs compacts than on the GP compacts at day 7. In vivo experiment showed that the MWNTs could induce ectopic bone formation in the dorsal musculature of ddy mice while GP could not. The results indicated that MWNTs might stimulate inducible cells in soft tissues to form inductive bone by concentrating more proteins, including bone-inducing proteins.