Time-dependent effects of ultraviolet and nonthermal atmospheric pressure plasma on the biological activity of titanium

Abstract

Here, we evaluated time-dependent changes in the effects of ultraviolet (UV) and nonthermal atmospheric pressure plasma (NTAPPJ) on the biological activity of titanium compared with that of untreated titanium. Grade IV machined surface titanium discs (12-mm diameter) were used immediately and stored up to 28 days after 15-min UV or 10-min NTAPPJ treatment. Changes of surface characteristics over time were evaluated using scanning electron microscopy, surface profiling, contact angle analysis, X-ray photoelectron spectroscopy, and surface zeta-potential. Changes in biological activity over time were as determined by analysing bovine serum albumin adsorption, MC3T3-E1 early adhesion and morphometry, and alkaline phosphatase (ALP) activity between groups. We found no differences in the effects of treatment on titanium between UV or NTAPPJ over time; both treatments resulted in changes from negatively charged hydrophobic (bioinert) to positively charged hydrophilic (bioactive) surfaces, allowing enhancement of albumin adsorption, osteoblastic cell attachment, and cytoskeleton development. Although this effect may not be prolonged for promotion of cell adhesion until 4 weeks, the effects were sufficient to maintain ALP activity after 7 days of incubation. This positive effect of UV and NTAPPJ treatment can enhance the biological activity of titanium over time.

Figure 1. Results of three-dimensional (3D) surface topographic analysis of the titanium disc surface immediately after UV and NTAPPJ compared with that of the control group.

Surface roughness parameters, Sa (A) and Sz (B), were quantitatively measured at a magnification of 10× with a scanning area of 310 μm × 230 μm, and the results were compared between groups.

Figure 2. Changes in the hydrophilicity of the titanium disc surface after treatment over time.

(A) Changes in wettability by water over time, as measured using a 4-μL H₂O droplet on the centre of each sample surface, between UV- and NTAPPJ-treated discs. (B) Changes in the contact angle with the titanium disc surface over time for the UV- and NTAPPJ-treated groups. Comparison of changes in the contact angle immediately (C) and 28 days (D) after treatment between groups. (E) Changes in the spread area of the titanium disc surface over time. Comparison of changes in the spread area immediately (F) and 28 days (G) after treatment between groups. ***P < 0.001 for comparisons between the indicated groups.

Figure 3. Changes in the chemical composition of the titanium disc surface after treatment over time.

Changes in Ti2p (A), O1s (B), and C1s (C) spectra immediately and 28 days after treatment between groups. (D) Changes in atomic percentages of carbon over time after treatment between groups.

Figure 4

(A) Changes in the zeta potential of the titanium disc surface after treatment over time at pH 7.4. Comparison of changes in zeta potential immediately (B) and 28 days (C) after treatment between groups. **P < 0.01, ***P < 0.001 for comparisons between the indicated groups.

Figure 5

(A) Changes in albumin adsorption rates on the titanium disc surface after treatment over time. Comparison of changes in albumin adsorption rates immediately (B) and 28 days (C) after treatment between groups. **P < 0.01, ***P < 0.001 for comparisons between the indicated groups.

Figure 6. Changes in relative osteoblastic cell attachment rates on the titanium disc surface after treatment over time.

Cell attachment rates immediately after treatment after 4 h (A) and 24 h (B) of incubation and 28 days after treatment after 4 h (C) and 24 h (D) of incubation. *P < 0.05, **P < 0.01 for comparisons between the indicated groups.

Figure 7. Changes in cellular morphology on the titanium disc surface after treatment over time.

Fluorescent stained (A) blue coloration for nuclei using DAPI and (B) red coloration for F-actin filaments using rhodamine phalloidin. (C) Comparison of cytoskeleton development, including area, perimeter, and Feret’s diameter of the cells, after treatment at each time point. ***P < 0.001 for comparisons between the indicated groups.

Figure 8

(A) Changes in ALP activity of the titanium disc surface after treatment over time, as determined by measuring the optical density (OD). Comparison of OD values immediately (B) and 28 days (C) after treatment between groups. *P < 0.05 for comparisons between the indicated groups.