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, 20 (23)

UV Treatment Improves the Biocompatibility and Antibacterial Properties of Crystallized Nanostructured Titanium Surface

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UV Treatment Improves the Biocompatibility and Antibacterial Properties of Crystallized Nanostructured Titanium Surface

Mai Hatoko et al. Int J Mol Sci.

Abstract

This study describes the production of a new material composed of pure titanium (Ti) metal with a crystallized nanostructure and investigated whether heat treatment and ultraviolet (UV) irradiation improved its biocompatibility and antibacterial properties. We compared the performance of UV-irradiated and non-irradiated Ti nanosheets (TNS) formed by dark alkaline treatment and heating at 600 °C with that of untreated pure Ti nanostructure (positive control). In vitro and in vivo experiments to assess biocompatibility and effects on cell behavior were performed using human umbilical vein endothelial cells and rat bone marrow cells. The material surface was characterized by X-ray photoelectron spectroscopy (XPS). The antibacterial properties of the irradiated material were evaluated using Staphylococcus aureus, a common pathogenic bacterium. The UV-irradiated TNS exhibited high angiogenic capacity and promoted cell adherence and differentiation relative to the control. Further, surface analysis via XPS revealed a lower C peak for the UV-treated material, indicating a reduced amount of dirt on the material surface. Moreover, UV irradiation decreased the viability of S. aureus on the material surface by stimulating reactive oxygen species production. The biocompatibility and antibacterial properties of the TNS were improved by UV irradiation. Thus, TNS may serve as a useful material for fabrication of dental implants.

Keywords: UV treatment; antibacterial; bone differentiation; crystallization; implant.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Surface analysis; (a) Scanning electron microscopy (SEM), (b) scanning probe microscopy (SPM), (c) X-ray photoelectron spectroscopy (XPS), (d) thin-film X-ray powder diffractometry (TF-XRD), (e) contact-angle.
Figure 2
Figure 2
The amount of bovine serum albumin (BSA) adsorbed on sample surfaces after 1, 3, 6, or 24 h of incubation was examined. TNS (Ti-nanosheet)-heat-UV showed the highest amount of protein adsorption, which increased in a time-dependent manner (* p < 0.05 vs. TNS, # p < 0.05 vs. TNS-heat).
Figure 3
Figure 3
(a) Phalloidin staining confirmed the adherence of cells and revealed the extension of cell processes in all groups, although the longest processes were observed in the TNS-heat-UV group; (b) Rat bone marrow (RBM) cell adhesion on the TNS disks was assessed after 1, 3, 6, or 24 h of culture. Adherence was highest in the TNS-heat-UV group at each time point; (c) After 7 and 14 days, alkaline phosphatase (ALP) activity was highest in the TNS-heat-UV group. (d,e) Further, an increase in the expression of osteocalcin (OCN), together with higher calcium deposition was observed in the TNS-heat-UV group relative to the TNS-heat and TNS control groups after 21 and 28 days (* p < 0.05 vs. TNS, # p < 0.05 vs. TNS-heat).
Figure 4
Figure 4
Osteogenesis-related genes including ALP, runt-related transcription factor (Runx2), bone morphogenetic protein (BMP)-2, and osteopontin (OPN) were upregulated in a time-dependent manner in RBM cells grown on different surfaces for 3, 7, 14, and 21 days, as determined by quantitative real-time PCR (qRT- PCR), with the highest levels observed in the TNS-heat-UV group (* p < 0.05 vs. TNS, # p < 0.05 vs. TNS-heat).
Figure 5
Figure 5
(a) TNS-heat-UV group showed the highest rate of adherence at each time point; (b) The adhesion of Human Umbilical Vein Endothelial Cells (HUVECs) on the Ti disks after 0.5, 1, and 3 h of culture; (c) The mRNA levels of intercellular adhesion molecule (ICAM)-1 and Von Willebrand factor were higher in the TNS-heat-UV and TNS-heat groups than in the control groups after 2 and 5 days of culture, respectively (* p < 0.05 vs. TNS, # p < 0.05 vs. TNS-heat).
Figure 6
Figure 6
The antibacterial potency of the TNS disks was evaluated using Staphylococcus aureus. The antibacterial rates were 0% for TNS-heat and 60% for TNS-heat-UV after 1 h, and 22% and 96%, respectively, after 6 h.
Figure 7
Figure 7
TNS, TNS-heat, and TNS-heat-UV disks were incubated with Staphylococcus aureus MG-1. Biofilm formation was evaluated by crystal violet staining after 18 and 24 h incubation. Biofilm formation was observed on TNS and TNS-heat but fewer on TNS-heat-UV after 18 and 24 h. (* p < 0.05 vs. TNS)
Figure 8
Figure 8
Live/dead and Reactive Oxygen Species (ROS) staining of the bacteria attached to sample disks. After 6 h of culture, bacteria grown on TNS and TNS-heat continued to proliferate until they covered the surface; 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining was negative (Figure 6). However, bacteria grown on TNS-heat-UV were positive for DCFH-DA after 1 and 6 h. Scale bar = 200 µm.
Figure 9
Figure 9
Micro-CT images (the implants were marked with red color, the cortical bone with blue color, and the cancellous bone with green color).
Figure 10
Figure 10
Quantitative evaluation of the trabecular bone within ROI (region of interest) (BV/TV, Tb.N, Tb.Th and Tb.Sp) * p > 0.05.

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