Multi-walled carbon nanotubes promote cementoblast differentiation and mineralization through the TGF-β/Smad signaling pathway

Abstract

Excretion of cementum by cementoblasts on the root surface is a process indispensable for the formation of a functional periodontal ligament. This study investigated whether carboxyl group-functionalized multi-walled carbon nanotubes (MWCNT-COOH) could enhance differentiation and mineralization of mammalian cementoblasts (OCCM-30) and the possible signaling pathway involved in this process. Cementoblasts were incubated with various doses of MWCNT-COOH suspension. Cell viability was detected, and a scanning electron microscopy (SEM) observed both the nanomaterials and the growth of cells cultured with the materials. Alizarin red staining was used to investigate the formation of calcium deposits. Real-time PCR and western blot were used to detect cementoblast differentiation and the underlying mechanisms through the expression of the osteogenic genes and the downstream effectors of the TGF-β/Smad signaling. The results showed that 5 µg/mL MWCNT-COOH had the most obvious effects on promoting differentiation without significant toxicity. Alp, Ocn, Bsp, Opn, Col1 and Runx2 gene expression was up-regulated. Smad2 and Smad3 mRNA was up-regulated, while Smad7 was first down-regulated on Day 3 and later up-regulated on Day 7. The elevated levels of phospho-Smad2/3 were also confirmed by western blot. In sum, the MWCNT-COOH promoted cementoblast differentiation and mineralization, at least partially, through interactions with the TGF-β/Smad pathway.

Figure 1

Cytotoxicity of carboxyl group-functionalized multi-walled carbon nanotubes (MWCNT-COOH) on cementoblasts at various concentrations after three days of incubation. Data are expressed as the mean ± SD. * p < 0.05, for 15 µg/mL MWCNT-COOH cells vs. control cells; ^a p < 0.001, for 25 µg/mL MWCNT-COOH cells vs. control cells.

Figure 2

Representative images showing the characteristic features of MWCNT-COOH (a) and cementoblasts cultured with it (b–d): (b) cells were able to adhere and spread well in all directions with normal morphology; (c) numerous filopodia and microvillosities were extended from the cells; (d) higher magnifications show cell attachment to clusters of MWCNT-COOH.

Figure 3

The alizarin red staining of the calcified nodules of cementoblasts after 14 days cultured in various concentrations of MWCNT-COOH: (a) 5 µg/mL; (b) 15 µg/mL; (c) 25 µg/mL; and (d) control.

Figure 4

Results of gene expression. (a) Expression of genes associated with differentiation and mineralization of cementoblasts exposed to various concentrations of MWCNT-COOH; (b) time-course of the gene expression of cementoblasts in response to 5 µg/mL MWCNT-COOH; (c) expression of the runt-related transcription factor 2 (Runx2) and Smad genes of cementoblasts in response to 5 µg/mL MWCNT-COOH. Data are shown as the mean ± SD (* p < 0.05; ** p < 0.001).

Figure 4

Results of gene expression. (a) Expression of genes associated with differentiation and mineralization of cementoblasts exposed to various concentrations of MWCNT-COOH; (b) time-course of the gene expression of cementoblasts in response to 5 µg/mL MWCNT-COOH; (c) expression of the runt-related transcription factor 2 (Runx2) and Smad genes of cementoblasts in response to 5 µg/mL MWCNT-COOH. Data are shown as the mean ± SD (* p < 0.05; ** p < 0.001).

Figure 5

Effects of MWCNT-COOH on Smad proteins by western blot. On Day 3, increased p-Smad2/3 was observed in cementoblasts treated with 5 µg/mL MWCNT-COOH compared to the control.