Osteoblastic Differentiation on Graphene Oxide-Functionalized Titanium Surfaces: An In Vitro Study

Abstract

Background: Titanium implant surfaces are continuously modified to improve biocompatibility and to promote osteointegration. Graphene oxide (GO) has been successfully used to ameliorate biomaterial performances, in terms of implant integration with host tissue. The aim of this study is to evaluate the Dental Pulp Stem Cells (DPSCs) viability, cytotoxic response, and osteogenic differentiation capability in the presence of GO-coated titanium surfaces.

Methods: Two titanium discs types, machined (control, Crtl) and sandblasted and acid-etched (test, Test) discs, were covalently functionalized with GO. The ability of the GO-functionalized substrates to allow the proliferation and differentiation of DPSCs, as well as their cytotoxic potential, were assessed.

Results: The functionalization procedures provide a homogeneous coating with GO of the titanium surface in both control and test substrates, with unchanged surface roughness with respect to the untreated surfaces. All samples show the deposition of extracellular matrix, more pronounced in the test and GO-functionalized test discs. GO-functionalized test samples evidenced a significant viability, with no cytotoxic response and a remarkable early stage proliferation of DPSCs cells, followed by their successful differentiation into osteoblasts.

Conclusions: The described protocol of GO-functionalization provides a novel not cytotoxic biomaterial that is able to stimulate cell viability and that better and more quickly induces osteogenic differentiation with respect to simple titanium discs. Our findings pave the way to exploit this GO-functionalization protocol for the production of novel dental implant materials that display improved integration with the host tissue.

Keywords: dental pulp stem cells; graphene oxide; osteoblastic differentiation; surface functionalization; titanium disc.

Figure 1

Raman spectra of control + GO (blue line), control (black dash dotted line), test + GO (red line), and test (green dash dotted line). Control + GO and test + GO spectra are normalized with respect to the G band.

Figure 2

Atomic force microscope (AFM) images of (A,D) bidimensional and (B,E) tridimensional topography, as well as (C,F) peak force error of control (upper line) and control + graphene oxide (GO) discs (lower line).

Figure 3

AFM images of (A,D) bidimensional and (B,E) tridimensional topography, as well as (C,F) peak force error of test (upper line) and test + GO discs (lower line).

Figure 4

SEM images of (A) control; (B) control + GO; (C) test; and (D) test + GO discs.

Figure 5

SEM images of DPSCs cultured on control, test, control + GO, and test + GO discs for 3, 14, and 28 days. Magnification 2000×.

Figure 6

3-[4,5-dimethyl-thiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT ) assay in DPSCs cultured on Ti discs for 3, 7, 14, 21, and 28 days. The histogram represents optical density (OD) values in control, test, control + GO, and test + GO discs. Data shown are the mean (± SD) of three separate experiments. * Day 7: test + GO vs. control, test, control + GO p < 0.01. * Day 14: test + GO vs. control, test p < 0.05. * Day 28: test vs. control p < 0.05.

Figure 7

LDH assay of DPSCs cultured on control, test, control + GO, and test + GO discs for 3, 7, 14, 21, and 28 days. LDH released is reported as percentage. Data shown are the mean (± SD) of three separate experiments.

Figure 8

Relative gene expression of (A) TGFβ and (B) BMP2 in DPSCs cultured on Ti discs for 1, 3, 7, 14, 21, and 28 days. Data are expressed as relative to control (calibrator sample, defined as 1). Values represent the means ± SD. Y-axis, fold change. The most representative of three separate experiments is shown.

Figure 9

Relative gene expression of (A) RUNX2, (B) SP7, and (C) COL1A1 in DPSCs cultured on Ti discs for 1, 3, 7, 14, 21, and 28 days. Data are expressed as relative to control (calibrator sample, defined as 1). Values represent the means ± SD. Y-axis, fold change. The most representative of three separate experiments is shown. *RUNX2, day 1: test + GO vs. control, test, control + GO p < 0.05; day 7: control + GO vs. control p < 0.05, test + GO vs. control p < 0.05; day 14: test + GO vs. control, test, control + GO p < 0.05. *SP7, day 7: control + GO, vs. control, test p < 0.01; test + GO vs. control, test p < 0.01; day 14: test + GO vs. control, test p < 0.01. *COL1A1, day 1: test + GO vs. control, test p < 0.01.

Figure 10

ELISA assay for PGE2 secretion of DPSCs cultured on Ti discs for 14, 21, and 28 days. Secretion levels are reported as picograms per milliliter per MTT optical density (OD) values. The results are the mean ± SD of three samples from three different experiments. * Day 21 and day 28: test + GO vs. control, test, control + GO p < 0.05.