Graphene Oxide Loaded on TiO2-Nanotube-Modified Ti Regulates the Behavior of Human Gingival Fibroblasts

Abstract

Surface topography, protein adsorption, and the loading of coating materials can affect soft tissue sealing. Graphene oxide (GO) is a promising candidate for improving material surface functionalization to facilitate soft tissue integration between cells and biomaterials. In this study, TiO₂ nanotubes (TNTs) were prepared by the anodization of Ti, and TNT-graphene oxide composites (TNT-GO) were prepared by subsequent electroplating. The aim of this study was to investigate the effect of TNTs and TNT-GO surface modifications on the behavior of human gingival fibroblasts (HGFs). Commercially pure Ti and TNTs were used as the control group, and the TNT-GO surface was used as the experimental group. Scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction were used to perform sample characterization. Cell adhesion, cell proliferation, cell immunofluorescence staining, a wound-healing assay, real-time reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blotting showed that the proliferation, adhesion, migration, and adhesion-related relative gene expression of HGFs on TNT-GO were significantly enhanced compared to the control groups, which may be mediated by the activation of integrin β1 and the MAPK-Erk1/2 pathway. Our findings suggest that the biological reactivity of HGFs can be enhanced by the TNT-GO surface, thereby improving the soft tissue sealing ability.

Keywords: MAPK pathway; graphene oxide; human gingival fibroblasts; soft tissue integration; titanium dioxide nanotubes.

Figure 1

Results of surface characterization of the Ti, TNT, and TNT-GO samples. (A) SEM images; (B) Raman spectra; (C) XRD patterns; (D) High—resolution XPS spectra of C 1s and O 1s. Abbreviations: Ti, pure titanium; TNT, TiO₂ nanotubes; TNT-GO, TiO₂ nanotubes with graphene oxide; XRD, X-ray diffraction; XPS, X-ray photoelectron spectroscopy. GO film was marked by red arrow.

Figure 2

Surface analysis of the Ti, TNT, and TNT-GO samples. (A) Reconstructed topographical images of the samples determined from atomic force microscopy; (B) water contact angles; (C) unbound and bound protein concentrations; (D) nanoscratch results for TNT and TNT-GO. Red arrow and force fluctuations indicate fracture at a displacement of 530 μm. (* p < 0.05).

Figure 3

(A) Cell adhesion of human gingival fibroblasts (HGFs) on the samples after 1, 2, and 4 h. (B) Cell proliferation of HGFs after 1, 3, 5, and 7 d. (C) DAPI staining of adherent HGFs after 1, 2, and 4 h. (* p < 0.05).

Figure 4

Scanning electron microscopy (SEM) images of HGFs on the different surfaces. (A) 1, 2, and 4 h. (B) 24 h. Red arrows indicate filopodia anchorage within nanotubes.

Figure 5

Immunofluorescence staining of HGFs on the different surfaces at 4 h and 24 h. F-actin was labeled red, and nuclei were labeled blue. (A) Vinculin (green); (B) integrin β1 (green). (C) Wound-healing assays at 0 and 24 h. Red arrow indicates punctate mature focal adhesion sites expressed at the edge of the pseudopodia.

Figure 6

Gene expression of HGFs on the different surfaces after (A) 4 h and (B) 24 h; (C) ELISA results of COL-1 and FN in HGFs at 1, 3, and 6 d; (D) Western blot of ERK and p-ERK at 4 h and 24 h (* p < 0.05).

Figure 7

Cell analysis at 1, 2, and 4 h after inhibiting the Erk1/2 signaling pathway. (A) Cell adhesion of HGFs; (B) DAPI staining of adherent HGFs; immunofluorescence staining of (C) vinculin and (D) integrin β1.

Figure 8

RT-PCR, ELISA, and Western blot results after inhibiting the Erk1/2 signaling pathway. (A) RT-PCR results of gene expression in HGFs at 24 h; (B) ELISA of COL-1 and FN in HGFs at 1, 3, and 6 d; (C) Western blot results of ERK and p-ERK at 24 h (* p < 0.05).