Enhancement of Biocompatibility of High-Transparency Zirconia Abutments with Human Gingival Fibroblasts via Cold Atmospheric Plasma Treatment: An In Vitro Study

Abstract

The objective of this study was to explore the effects of cold atmospheric plasma (CAP) treatment on the biological behavior of human gingival fibroblasts (HGFs) cultured on the surface of high-transparency zirconia. Two types of zirconia, 3Y-ZTP and 4Y-PSZ, were subjected to a CAP treatment for various treatment durations. Analyses of the physical and chemical properties of 3Y-ZTP and 4Y-PSZ were conducted using scanning electron microscopy, contact angle measurements, and X-ray photoelectron spectroscopy, both before and after CAP treatment. The biological responses of HGFs on both surfaces were assessed using CCK-8 assay, confocal laser scanning microscopy, and real-time PCR. Initially, the oxygen and hydroxyl contents on the surface of 4Y-PSZ exceeded those on 3Y-ZTP. CAP treatment enhanced the surface hydrophilicity and the reactive oxygen species (ROS) content of 4Y-PSZ, while not altering the surface morphology. After CAP treatment, HGFs' adhesion on 4Y-PSZ was superior, with more pronounced effects compared to 3Y-ZTP. Notably, HGFs counts and the expression of adhesion-related genes on 4Y-PSZ peaked following the CAP exposures for 30 s and 60 s. Consequently, this study demonstrates that, following identical CAP treatments, 4Y-PSZ is more effective in promoting HGFs adhesion compared to traditional 3Y-ZTP zirconia.

Keywords: abutment surface modification; cold atmosphere plasma; high-transparency zirconia; reactive oxygen species.

Figure 1

Surface morphology of 4Y-PSZ as SEM images. (a) 4Y-PSZ-0 s and (b) 4Y-PSZ-90 s (500 magnification).

Figure 2

Surface wettability of 3Y-ZTP and 4Y-PSZ specimens at the same CAP treatment times (n = 12). (** p < 0.01; * p < 0.05).

Figure 3

High-resolution XPS spectra of 3Y-ZTP and 4Y-PSZ surfaces. O 1 s high-resolution image of 3Y-ZTP and 4Y-PSZ. (a) 3Y-ZTP-0 s; (b) 3Y-ZTP-30 s; (c) 3Y-ZTP-60 s; (d) 3Y-ZTP-90 s; (e) 4Y-PSZ-0 s; (f) 4Y-PSZ-30 s; (g) 4Y-PSZ-60 s; and (h) 4Y-PSZ-90 s.

Figure 4

Observation of HGFs on 3Y-ZTP and 4Y-PSZ Surface by Confocal Laser Scanning Microscope. Confocal laser scanning microscopy images of HGFs on zirconia disks at 3 h (A,B) and 24 h (C,D) of culture. The CAP treatment times are 0 s (a,e), 30 s (b,f), 60 s (c,g), and 90 s (d,h). Low magnification (a–d), scale bar = 20 μm. High magnification (e–h), scale bar = 20 μm.

Figure 4

Observation of HGFs on 3Y-ZTP and 4Y-PSZ Surface by Confocal Laser Scanning Microscope. Confocal laser scanning microscopy images of HGFs on zirconia disks at 3 h (A,B) and 24 h (C,D) of culture. The CAP treatment times are 0 s (a,e), 30 s (b,f), 60 s (c,g), and 90 s (d,h). Low magnification (a–d), scale bar = 20 μm. High magnification (e–h), scale bar = 20 μm.

Figure 5

Perimeters and spreading areas of the HGFs on 4Y-PSZ surfaces. Perimeters (a,b) and spreading areas (c,d) of the HGFs on 4Y-PSZ surfaces at 3 h (a,c) and 24 h (b,d). Data are presented as means ± SD (n = 6). Identical letters indicate that the differences between those values have no sta-tistical significance (p > 0.05).

Figure 6

Comparison of HGFs’ perimeters and spreading areas on 3Y-ZTP and 4Y-PSZ surfaces at the same CAP treatment times. Perimeters (a,b) and spreading areas (c,d) of the HGFs on 3Y-ZTP and 4Y-PSZ surfaces at 3 h (a,c) and 24 h (b,d). Data are presented as means ± SD (n = 6). (** p < 0.01; * p < 0.05).

Figure 7

Quantitative measurements of the HGFs on 4Y-PSZ surfaces. HGFs’ attachment after culturing for (a) 3 h and (b) 24 h and proliferation after culturing for (c) 48 h and (d) 72 h. Data are presented as means ± SD (n = 12). Identical letters indicate that the differences between those values have no statistical significance (p > 0.05).

Figure 8

Quantitative comparison of HGFs on 3Y-ZTP and 4Y-PSZ surfaces at the same CAP treatment times (n = 12). CAP treatment times: (a) 0 s, (b) 30 s, (c) 60 s, and (d) 90 s (p > 0.05). (** p < 0.01; * p < 0.05).

Figure 9

Analyses on the expressions of genes involved in the HGFs’ adhesion on 4Y-PSZ. Analysis of the expression of (a) focal adhesion kinase, (b) fibronectin, (c) integrin β3, and (d) vinculin after their culture in real-time PCR for 24 h. Data represent fold changes of the target genes relative to the GAPDH expression and HGFs grown in a control group (100%). The values are presented as means ± SD. Identical letters indicate that the differences between those values have no statistical significance (p > 0.05).

Figure 10

Comparison of expressions of genes involved in the HGFs’ adhesion on 3Y-ZTP and 4Y-PSZ surfaces at the same CAP treatment times (n = 12). CAP treatment times: (a) 0 s, (b) 30 s, (c) 60 s, and (d) 90 s (p > 0.05). (** p < 0.01; * p < 0.05).