Open air atmospheric pressure plasma jet (APPJ) enhanced chemical vapour deposition process was used to deposit biocompatible SiOx/TiOx composite coatings. The as deposited films are hydrophilic and show visible light induced photocatalytic effect, which is a consequence of the formation of defects in the TiOx structure due to the plasma process. This photocatalytic effect was verified by the demonstration of an antimicrobial effect under visible light on E. coli as well as by degradation of Rhodamine B. The films are non-cytotoxic as shown by the cytocompatibility tests. The films are conductive to cell growth and are stable in DMEM and isopropanol. The structural evaluation using SEM, EDS and XPS shows a dispersion of TiOx phase in a SiOxCyHz matrix. These analyses were used to correlate the structure-property relationship of the composite coating.
Plasma deposited titanium oxide coatings (TiOx) are widely used and investigated in electrical and optical applications . In addition, due to its biocompatibility and antimicrobial properties, it is an interesting material for implants . However, a small drawback is its bioinert nature . To overcome this, possible strategies are to optimize the topography or to mix it with another material that can activate cell growth . Silicon oxide supports cell growth and hence can be used as the other phase in the mixture . Another advantage of adding silicon oxide would be the improvement in mechanical stability and abrasive-wear resistance of the coating.
To generate composite SiOx/TiOx coatings various processes are used, e.g. Sol-Gel process , ,  and plasma-enhanced chemical vapour deposition (PE-CVD) . The Sol-Gel process is a wet chemical process and the PE-CVD is commonly performed under vacuum. To eliminate the chemical waste and the expensive vacuum equipment an APPJ can be used alternatively. The APPJ operates in ambient conditions, the plasma parameters are relatively easy to control, and it is effortless implementable in an in-line process .
Another reason for using APPJ process for titanium oxide coating is the modifying effect on the photocatalytic properties of the TiOx coating. Typically, pure titanium oxide is activated by UV light, and only through doping a redshift of this effect to visible light can be induced. In principle, the presence of dopants reduce the band gap and hence redshift the effect. This redshift was observed, by Fakhouri et al. , in the absence of dopants in visible light, however only after a thermal post treatment of the APPJ deposited titanium oxide films. This was verified by evaluating the antimicrobial effect induced by visible light in absence of dopants, and by degradation of Rhodamine B under UV light. The advantage of this coating is that it can be reactivated under normal light, indoors or outdoors, compared to a classical material that would need a UV lamp.
In this work we have demonstrated the creation of SiOx/TiOx composite coatings that are hydrophilic, mechanically stable, biocompatible and antimicrobial, induced by visible light, with an APPJ process without any post- treatment.
2 Material and methods
2.1 Preparation of SiOx/TiOx thin films
The standard industrial PlasmaPlus® system from Plasmatreat GmbH (see Figure 1) was used to deposit the coatings. Clean dry industrial air with a flow rate of 33 slm was used as ionisation gas in the plasma torch, working at 19 kHz and 280 V. The precursor, a solution of titanium (IV) isopropoxide (TTIP) and tetraethyl orthosilicate (TEOS) was mixed at 40 g/h in an evaporator at 240°C with 2 slm nitrogen which played the role of a carrier gas to bring the precursor to the plasma. Stainless steel 316L plates (1 cm × 1 cm × 0.1 cm) were used as the substrate. The substrates were cleaned with ethanol and acetone in an ultrasonic bath and afterwards etched with HNO3 (65%) for 10 min. They were then pre-treated with two 10 m/min passes of open air plasma at a nozzle to sample distance of 20 mm. Finally, they were coated for 3 s by adding the precursor into the plasma. The resulting coatings were optically visible and created a light yellow shining on the surface.
2.2 Characterization of the films
2.2.1 Structural Characterization
The chemical composition of the samples was studied by XPS (Leybold-Heraeus, LHS10). The measurements were performed using a MgKα X-ray source (λ = 1253.6 eV) at a pressure of 3 × 10−8 mbar. The surface morphology study and a semi quantitative chemical composition analysis was performed using SEM-EDS (JEOL Ltd., JSM-6060LV). The surface was also characterized using a contact angle measuring system (DataPhysics Instruments GmbH, OCA20) with distilled water to evaluate the contacusingt angle. Stability was tested by immersing the samples in i) water, ii) Dulbecco’s Modified Eagle Medium (DMEM) and iii) Isopropanol, for 7 days and evaluating the structural changes using SEM measurements.
2.2.2 Cytotoxicity evaluation
The cytotoxicity of the samples was tested according to DIN EN ISO 10993-5. Eluate tests were performed with a fibroblast cell line (Hs27) and a cell counting test (Dojindo, CCK-8). To further characterize the biological properties of the film, two more tests were performed: i) direct Hs27 colonization of the samples following CCK-8 assay, ii) direct Hs27 colonization, fixation and drying of the cells for SEM studies.
2.2.3 Anti-microbial effect determination
The antimicrobial effect was determined by degradation of Rhodamine B under UV light. To evaluate the reaction towards bacteria the samples were inoculated with E. coli and irradiated for 1 h with visible light. The samples were then washed with PBS and the wash solution was put on agar plates and incubated at 37°C for 24 h. The colony building units were counted and compared with the non-irradiated control sample.
3 Results and discussion
3.1 Surface composition, wettability and morphology
The SEM studies showed a rough granular surface based on nanoparticles. By analysing the surface with EDS, the surface shows titanium and silicon distributed on the surface (see Figure 2). The grains of the steel are still visible but the coating is homogeneous and dense. The mean size of the particles is around 500 nm ranging from around 185 nm to 856 nm.
The XPS spectroscopy gave an overview of the composition of the films. The titanium as well as the silicon peak was clearly visible, and by fitting the curves the following bonds could be detected: Ti-OH, TiO2, SiO2, Si-C. If the results are analysed in detail, one may come to the conclusion that there is a phase separation between the two SiOx and TiOx type of materials. A dispersed TiO based phase in a SiOC matrix could be interpreted.
Contact angle measurement showed a contact angle of 20° 1 day after film deposition, demonstrating that the surface is hydrophilic. It is well known that titanium oxide surfaces are superhydrophilic and silicon oxide surfaces exhibits contact angles of near 80° with water. Therefore, as presumed these composite films exhibit an intermediate contact angle.
3.2 Cellular reaction
In the eluate test the CCK-8 assay showed no cytotoxicity of the films, since reduction of proliferation has to be below 70% to be classified as cytotoxic (see Figure 3). Copper was used as positive and silicone as negative control.
All results are evaluated in comparison to a reference (cells in a cell culture plate). Cells on the tested films show a higher proliferation than on stainless steel, which means no toxic components are introduced into the cell culture medium, rather the opposite. This behaviour may be attributed to the presence of silicon oxide that enhances the cell growth .
SEM analysis after direct colonization of Hs27 on the surface showed, that the cells adhere and spread on the surface (see Figure 4).
The direct proliferation test showed a lower value for the coated surface as for stainless steel. All results are evaluated in comparison to the control. For the film the proliferation is around 75% and in comparison the one of stainless steel is around 100%. This could be due to the surface morphology. It is known that granular surfaces may hinder fibroblast attachment and growth.
The results indicate, that the films can be used in contact with human tissue due to the non-cytotoxicity and adhesion properties for cells.
3.3 Antimicrobial behaviour of the films
The photocatalytic effect, which is the basis for antimicrobial properties, was determined by measuring the degradation of Rhodamine B under UV light (365 nm). Over time a decrease of the mean extinction can be measured (see Figure 5). Titanium oxide surface exhibits photocatalytic activity by creation of electron hole pair through absorption of photons, corresponding to the energies higher than the band gap. These electron hole pairs can then create radicals which play a role in further reactions such as microorganism mortification and hence antimicrobial properties.
Samples inoculated with E. coli were irradiated under visible light (Osram T8 L 15W/840) for 1 h. Interestingly, a two log decrease in the bacteria colony concentration was observed. This indicated that the films have an antimicrobial effect induced by visible light.
The antimicrobial effect under UV-light is a well-known effect of titanium oxide, and only by introduction of dopants, one can redshift the band gap and hence the effect can be induced by visible light. However, the antimicrobial effect under visible light, in the present study, is a direct result of the atmospheric plasma process, which seems to result in the introduction of redshifting structural defects. Although this mechanism needs further evaluation, nevertheless, this renders this APPJ deposition technology extremely interesting.
3.4 Antimicrobial behaviour of the films
To test the suitability of the films for use on implants, immersion test were performed. The samples were immersed for 7 days in DMEM, distilled water and isopropanol (see Figure 6).
The coatings showed a good stability against isopropanol and DMEM. However, a solubility of the film was observed in water. To improve the water resistance the steel was sandblasted before the mentioned pre-treatment and coating steps. This led to a better resistance in water.
The APPJ deposited SiOx/TiOx composite film, with a distributed TiOx phase in a SiOx matrix, is mechanically stable and shows an antimicrobial effect under visible light without any post treatment. This makes it usable in everyday environment. The film is also biocompatible and stable in biological fluids and disinfectants making it usable for implant applications. Thus, the use of the APPJ process, to realize biocompatible and bioactive material surfaces holds a promising future to biological and medical applications
The authors are grateful to Christian Buske and Plasmatreat GmbH for their support.
Research funding: The author state no funding involved. Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animal use.
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