Christin Rapp, Andreas Baumgärtel, Lucas Artmann, Markus Eblenkamp and Syed Salman Asad

Open air plasma deposited antimicrobial SiOx/TiOx composite films for biomedical applications

Open Access
De Gruyter | Published online: September 30, 2016

Abstract

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.

1 Introduction

Plasma deposited titanium oxide coatings (TiOx) are widely used and investigated in electrical and optical applications [1]. In addition, due to its biocompatibility and antimicrobial properties, it is an interesting material for implants [2]. However, a small drawback is its bioinert nature [3]. To overcome this, possible strategies are to optimize the topography or to mix it with another material that can activate cell growth [4]. Silicon oxide supports cell growth and hence can be used as the other phase in the mixture [5]. 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 [6], [7], [8] and plasma-enhanced chemical vapour deposition (PE-CVD) [9]. 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 [10].

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. [11], 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.

Figure 1: Principal of an APPJ of the company Plasmatreat.

Figure 1:

Principal of an APPJ of the company Plasmatreat.

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.

Figure 2: SEM picture and measurement with EDS to demonstrate the distribution of titanium and silicon in the film.

Figure 2:

SEM picture and measurement with EDS to demonstrate the distribution of titanium and silicon in the film.

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.

Figure 3: CCK-8 assay for the film (SiOx/TiOx), copper, silicone and stainless steel (n = 5).

Figure 3:

CCK-8 assay for the film (SiOx/TiOx), copper, silicone and stainless steel (n = 5).

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 [6].

SEM analysis after direct colonization of Hs27 on the surface showed, that the cells adhere and spread on the surface (see Figure 4).

Figure 4: Fixed and dried fibroblasts on the film, examined by SEM.

Figure 4:

Fixed and dried fibroblasts on the film, examined by SEM.

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.

Figure 5: Degradation of Rhodamine B on the samples under UV light over time (n = 5, i = 3).

Figure 5:

Degradation of Rhodamine B on the samples under UV light over time (n = 5, i = 3).

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).

Figure 6: SEM pictures of the coating before and after immersion in DMEM, distilled water and isopropanol.

Figure 6:

SEM pictures of the coating before and after immersion in DMEM, distilled water and isopropanol.

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.

4 Conclusion

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

Acknowledgement

The authors are grateful to Christian Buske and Plasmatreat GmbH for their support.

Author’s Statement

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.

References

[1] Wang XY, Liu Z, Liao H, Klein D, Coddet C. Microstructure and electrical properties of plasma sprayed porous TiO2 coatings containing anatas. Thin Solid Films. 2004;451–452:37–42. Search in Google Scholar

[2] Sabetrasekh R, Tiainen H, Lyngstadaas SP, Reseland J, Haugen H. A novel ultra-porous titanium dioxide ceramic with excellent biocompatibility. J Biomater Appl. 2011;25:559–80. Search in Google Scholar

[3] Zhao X, You J, Xie Y, Cao H, Liu X. Nanoporous SiO2/TiO2 composite coating for orthopedic application. Matter Lett. 2015;152:1–4. Search in Google Scholar

[4] Sjöströma T, Dalby MJ, Hart A, Tare R, Oreffo RO, Su B. Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells. Acta Biomaterialia 2009;5:1433–41. Search in Google Scholar

[5] Beck GR, Ha SW, Camalier CE, Yamaguchi M, Li Y, Lee JK, et al. Bioactive silica-based nanoparticles stimulate bone-forming osteoblasts, suppress bone-resorbing osteoclasts, and enhance bone mineral density in vivo. Nanomedicine. 2012;8:793–803. Search in Google Scholar

[6] Ääritalo V, Areva S, Jokinen M, Lindén M, Peltola T. Sol-gel-derived TiO2-SiO2 implant coatings for direct tissue attachment. Part I: design, preparation and characterization. J Mater Sci: Mater Med. 2007;18:1863–73. Search in Google Scholar

[7] Areva S, Aäritalo V, Tuusa S, Jokinen M, Lindén M, Peltola T. Sol-gel-derived TiO2-SiO2 implant coatings for direct tissue attachment. Part II: evaluation of cell response. J Mater Sci: Mater Med. 2007;18:1633–42. Search in Google Scholar

[8] Yang L-L, Lai Y-S, Chen JS, Tsai PH, Chen PL, Jason Chang C. Compositional tailored sol-gel SiO2-TiO2 thin films: Crystallization, chemical bonding configuration, and optical properties. J Mater Res. 2005;20:3141–9. Search in Google Scholar

[9] Gracia F, Yubero F, Holgado JP, Espinos JP, Gonzalez-Elipe AR, Girardeau T. SiO2/TiO2 thin films with variable refractive index prepared by ion beam induced and plasma enhanced chemical vapor deposition. Thin Solid Films. 2006;500:19–26. Search in Google Scholar

[10] Merche D, Vandencasteele N, ReniersF. Atmospheric plasma for thin film deposition: A critical review. Thin Solid Films. 2012;520:4219–36. Search in Google Scholar

[11] Fakhouri H, Ben Salem D, Carton O, Pulpytel J, Arefi-Khonsari F. Highly efficient photocatalytic TiO2 coatings deposited by open air atmospheric pressure plasma jet with aerosolized TTIP precursor. J Phys D: Appl Phys. 2014;47:1–11. Search in Google Scholar

Published Online: 2016-9-30
Published in Print: 2016-9-1

©2016 Christin Rapp et al., licensee De Gruyter.

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