Effect of Hydrogen Peroxide on the Surface and Attractiveness of Various Zirconia Implant Materials on Human Osteoblasts: An In Vitro Study

Abstract

The aim of this in vitro study was to investigate the effect of hydrogen peroxide (H₂O₂) on the surface properties of various zirconia-based dental implant materials and the response of human alveolar bone osteoblasts. For this purpose, discs of two zirconia-based materials with smooth and roughened surfaces were immersed in 20% H₂O₂ for two hours. Scanning electron and atomic force microscopy showed no topographic changes after H₂O₂-treatment. Contact angle measurements (1), X-ray photoelectron spectroscopy (2) and X-ray diffraction (3) indicated that H₂O₂-treated surfaces (1) increased in hydrophilicity (p < 0.05) and (2) on three surfaces the carbon content decreased (33-60%), while (3) the monoclinic phase increased on all surfaces. Immunofluorescence analysis of the cell area and DNA-quantification and alkaline phosphatase activity revealed no effect of H₂O₂-treatment on cell behavior. Proliferation activity was significantly higher on three of the four untreated surfaces, especially on the smooth surfaces (p < 0.05). Within the limitations of this study, it can be concluded that exposure of zirconia surfaces to 20% H₂O₂ for 2 h increases the wettability of the surfaces, but also seems to increase the monoclinic phase, especially on roughened surfaces, which can be considered detrimental to material stability. Moreover, the H₂O₂-treatment has no influence on osteoblast behavior.

Keywords: cell culture; dental implant surface; hydrogen peroxide; osseointegration; primary human alveolar bone-derived osteoblasts; zirconia.

Figure 1

(A–C) Morphology of the zirconia-based surfaces before and after H₂O₂-treatment and changes in their hydrophilic properties after H₂O₂-treatment. (A) SEM images at 5000× magnification of the four untreated and H₂O₂-treated surfaces Zr1-m/r and Zr2-m/r (mean average roughness data ± SD (Ra in µm) given below SEM images). (B) Representative photographic images of contact angle measurements of 1 µL water droplets pipetted onto zirconia discs with and without H₂O₂-treatment for 2 h (mean contact angles ± are embedded in the contact angle images). (C) Comparison of the contact angle development with and without H₂O₂-treatment between the four different samples. Statistical significances with p < 0.05 are labeled with “*” in the graphs.

Figure 2

(A–D) XPS detail spectra of the smooth and roughened surfaces of the two zirconia materials (Zr1-m/r and Zr2-m/r) and changes in the chemical composition after H₂O₂-treatment for the following electrons: (A) = C1s; (B) = O1s; (C) = Zr3d. (D) XRD graphs of the four surfaces (a/b: Zr1-m/r and c/d: Zr2-m/r) (t = tetragonal ZrO₂, m = monoclinic ZrO₂; c = cubic phase). Typical peaks of the monoclinic ZrO₂ phase at 2θ of 28.3° and 31.5° and peaks of the tetragonal/cubic phase at 2θ of 30.3° were detected on all surfaces. Additional specific peaks for the tetragonal phase could be identified, but they overlapped the cubic phase peaks. The surface of the Zr1 material showed several additional peaks indicative of additional crystalline structures.

Figure 3

(A,B) Initial attachment and spread of AO 4 h (A) and 24 h (B) after seeding onto untreated and H₂O₂-treated zirconia surfaces. Representative fluorescence microscopy images of cell cultures with dual staining of DAPI for nuclei (blue) and phalloidin for actin filaments (green) are shown.

Figure 4

(A,B) Comparison of the cell area (A) and the DNA concentration (B) on untreated and H₂O₂-treated zirconia surfaces after 4 h and 24 h of culture. The image analyzer tool of the fluorescence microscope was used to perform cell morphometric measurements. Data are mean ± SD (n = 81) evaluated from 27 cells from each disc. Statistical significances with p < 0.05 are labeled with “*” in the graphs.

Figure 5

(A,B) Proliferation and differentiation activity of AO on H₂O₂-treated and untreated surfaces. (A) Cell proliferation by the alamarBlue^® metabolic assay at 3, 7 and 14 days of culture. For a clear illustration of the development of proliferation rates, the graphs are labeled with the most important percentages at days 3, 7 and 14. (B) Determination of differentiation capacity by measuring alkaline phosphatase activity of the normalized protein amount (5 µg protein for each reaction) between H₂O₂-treated and untreated groups at days 7 and 14. The data were collected from triplicates of three independent experiments (n = 9), with a predefined constant cell number of 5 × 10⁴ cells per disc. Statistical significances with p < 0.05 are labeled with “*” in the graphs.