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Copyright V © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc. Copyright C 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Thoughts and Progress Influence of Oxynitrided Surface in the Production of a Less Susceptible Titanium Surface to Skin-Borne Bacterial Adhesion *Michelle de Medeiros Aires, †Janine Treter, *Danilo Cavalcante Braz, ‡Cristiano Krug, †Alexandre José Macedo, and *Clodomiro Alves Júnior *Lab Plasma, Federal University of Rio Grande do Norte, Rio Grande do Norte; †Biofilms and Microbial Diversity Laboratory, Biotechnology Center and Pharmacy College of the Federal University of Rio Grande do Sul; and ‡Physical Institute, Federal University of Rio Grande do Sul, Rio Grande do Sul, Brazil Abstract: There is a growing quest for an ideal biomaterial that shows appropriate cellular response and is not susceptible to microbial adhesion. In this study, commercial grade II titanium was submitted to RF/DC plasma surface modification at 2.2 mbar, using gas mixtures of argon, nitrogen, and oxygen at proportions 4:1:2 and 4:1:3. The surfaces were physically and chemically characterized. In order to evaluate bacterial response, the surfaces were exposed to Staphylococcus epidermidis. Oxynitrided samples, although having a higher roughness as compared with untreated samples, exhibited lower bacterial growth. This observation is probably due to the formation of different crystalline phases of nitrides and oxides caused by plasma treatment. The surface with highest contact angle and highest surface tension showed lower bacterial adhesion. These results were confirmed by scanning electron microscopy. The role of nitrogen in reducing bacterial adhesion is clear when this material is compared with untreated titanium, on which only an oxide film is present. Key Words: Plasma— Staphylococcus epidermidis—Biofilm—Titanium.
The increasing demand for implants and medical devices has led to a concomitant increase in the incidence of device-related infections (1). It is estimated that approximately 65% of hospital-acquired infections are related to surface-attached microorganisms (2). When any biomaterial is implanted, it can doi:10.1111/aor.12581 Received February 2015; revised June 2015. Address correspondence and reprint requests to Miss Michelle de Medeiros Aires, LabPlasma, Universidade Federal do Rio Grande do Norte, 59072-970 Rio Grande do Norte, Brazil. E-mail: [email protected]
become a site for bacterial adhesion, colonization, and infection (3). Bacteria tend to adhere to different types of surfaces, ranging from surfaces in the human body, and plants and clays, to plastic and metals (4). Among the metals, titanium and its alloys have increased their use as biomaterial stems, mainly in orthopedic and dental implants (5) due to their mechanical strength, biocompatibility, and corrosion resistance (6). The chemical and physical stability of titanium dioxide, TiO2, led to an increased attention toward this titanium compound in the last decade (7). The surface energy, thickness, roughness, and crystalline properties of this material are widely studied (8). It is well established in literature that titanium biocompatibility is connected with a nano-TiO2 layer on its surface (9). Intermediate stoichiometric titanium compounds (TiNxOy) began to be studied due to the ability of the nitrogen to act as a dopant in TiO2. The properties of the compound are strongly dependent on the ratio oxygen/nitrogen (10). Characteristics such as changing gap, resistivity, surface electron acceptors, or donors affect bacterial adherence on the surface. Staphylococcus epidermidis adheres minimally to the TiNxOy films deposited by physical vapor deposition with specific values of resistivity (11). It was found that the load transfer between TiNxOy surfaces and bacteria during the adhesion process depends upon the substrate specific resistivity and provides a strong influence on bacterial adhesion (12). Once the bacteria are adhered to the medical device, they start a multifactor process of biofilm formation. Biofilms are a protected mode of life of bacteria, where they are adhered on a surface embedded in a self-produced matrix (13), becoming more resistant to antibiotics and the host immune system (14). Most biomaterial-related infections are caused by skin commensals, in particular the coagulasenegative staphylococci (15) like S. epidermidis (16). To minimize microbial adhesion on titanium surfaces, several approaches have been made in recent years. However, antibacterial coatings are a complex issue, as they should be tailored to specific bacterial species (17). Some examples of attempts include antibiotic-loaded coatings (18), organic bactericide coatings (19), and inorganic bactericide surfaces like silver coating (20). ArtificialOrgans Organs2016, 2015,40(5):521–526 ••(••):••–•• Artificial
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It is well known that initial steps of biofilm formation are closely related to bacterial adhesion on the surface of the material, which is crucial for the success of bacterial colonization. Therefore, new strategies focus on changing physicochemical properties of the surfaces. These methods are relatively simple and an economic way to repel bacteria colonization (17). Among the different surface modification methods available, those that use plasma as a source of energy advanced considerably in recent years, mainly due to their versatility (6,21). Moreover, bacterial adhesion reduction on medical devices by a process without any drug incorporation is an important strategy to avoid nosocomial infections (22). In this work, modification of titanium surface by plasma was evaluated as a means of avoiding adhesion of the pathogenic skin-borne bacteria— S. epidermidis. The surface modification procedure was performed by using different gas compositions, where the influence of that parameter was carefully studied. MATERIALS AND METHODS Material and plasma treatment Titanium samples were prepared from Ti cp (Degree II). They were cut in a 9 mm × 3 mm (diameter × thickness) disk format. Initially, the titanium disks were sanded with silicon carbide sandpaper and polished by a porous neoprene (PANTEC, Zwick Roell AG, Ulm, Germany) impregnated with colloidal silica and hydrogen peroxide mixture solution for a 30-min period. The samples were afterwards washed with enzymatic detergent followed by absolute ethanol and distilled water. The samples were kept under ultrasonic agitation for 10 min at every step (23). Polished titanium disks without plasma treatment were used as reference in each assay. Surface modification of titanium disks was carried out in a cylindrical (300 mm × 300 mm) plasma reactor made of stainless steel, equipped with a direct current power supply and a vacuum system as previously described (24). Before the treatment of the disks was carried out, a precleaning step was performed in order to remove oxides and other contaminants using ionic bombardment. For this step, the following parameters were used: 2.0 sccm argon and 2.0 sccm hydrogen flow rates, 1.5 mbar pressure, 200°C temperature, and 594 V voltage. These conditions allowed formation of plasma, which contained neutral species as well as charged ones, such as Ar1+, H1+, and H2+. These species bombed and pulled out material at the surface of the sample. This sputtering and chemical erosion step was maintained for 30 min. Artif Organs, Vol. 2015 Vol. ••, 40, No. No. ••, 5, 2016
For plasma treatment itself, three different atmosphere conditions were used, where only the flow of oxygen was varied. The conditions employed were 4.0 sccm Ar, 1.0 sccm N2, and 2.0 or 3.0 sccm O2.The working pressure was set at 2.2 mbar and temperature during treatment was kept constant at 500°C for 1 h. Afterwards, the equipment was turned off and the samples were allowed to cool down in the reactor chamber. Surface characterization X-ray diffraction (XDR) measurements were carried out at grazing angle of 1° using the Shimadzu6000 diffractometer (Shimadzu Corporation, Kyoto, Japan), Cu Kα radiation at a 2θ scan from 25.0 to 45.0°. The surface topography and roughness of untreated and plasma-treated disks were measured with an atomic force microscope (AFM) (SPM9600, Shimadzu). Four roughness parameters were evaluated: average roughness (Ra), average maximum height of the profile (Rz), maximum profile peak height (Rp), and maximum profile valley depth (Rv). Nanotopography images were taken in three different regions of the sample. In each 2D image, lines were obtained for determination of roughness profiles. Surface wettability measurements were also conducted. A precleaning procedure using 0.5% of enzymatic detergent in double-distilled water followed by an ultrasonic treatment for 10 min was performed. The disks (untreated and plasma treated) were washed with absolute ethanol and subsequently with distilled water. The sessile drop method was employed to measure the static contact angles of different solvents on titanium disks (25). Doubledistilled water and glycerol (Vetec Quimica, Rio de Janeiro, Brazil) were used as previously described (24). The contact angle measurements were executed by dripping 20 μL of the solvent on the sample surface using a custom-made device (23). An average value of the contact angle was obtained from at least six measurements for each solvent/titanium surface combination. Images were taken at different times until complete relaxation of the drop with Pinnacle Studio 9.0 program (Corel Corporation, Ottawa, Canada). The measurement of surface tension was based on the drop geometric method developed by Fowkes (26). Microorganisms, culture conditions, and sample preparation Staphylococcus epidermidis ATCC 35984 cells were grown aerobically at 37°C for 24 h on MuellerHinton agar plates (OXOID) from frozen stocks. Several colonies were used to make the bacterial
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FIG. 1. X-Ray diffractogram in GIXRD of titanium disk samples: (a) untreated and (b) 4Ar–1N2–2O2 in solid line and 4Ar–1N2– 3O2 in dotted line.
suspension in sterile sodium chloride 0.9% at a final concentration of 3 × 108 CFU per mL. Biofilm formation method (27) was adapted to 24-well plates containing a sterile titanium disk. Briefly, in each well 400 μL of the bacterial suspension and 600 μL of tryptone soy broth were added (TSB) (OXOID). After incubation period of 6 and 24 h at 37°C, the titanium samples were washed twice with sterile 0.9% sodium chloride to remove free bacterial cells and fixed for 2 h in glutaraldehyde 2.5%. Then the samples were washed with 100 mM of sodium cacodylate buffer (pH 7.2) and treated for 2 h with 2% osmium tetroxide. After that, the samples were again washed with 100 mM of sodium cacodylate buffer (pH 7.2) and finally dehydrated using increasing concentrations of acetone. The dehydrated titanium disks were dried by the CO2 critical point technique (Balzers CPD 030, Leica Microsystems GmbH, Wetzlar, Germany), fixed on aluminum stubs, covered with a gold film, and examined in a JEOL JSM-6060 scanning electron microscope (JEOL USA, Inc., Peabody, MA, USA). RESULTS AND DISCUSSION Figure 1 shows the XRD analysis of untreated and treated samples with different O2 flow rates. The
results demonstrate the formation of different crystalline phases of oxides and nitrides on the titanium surface, such as TiO6, TiO2, Ti3N2x, and Ti4N3-x. The presence of TiO2 phase is observed at 27°, where the peak intensity increases with increasing oxygen gas flux in the treatments. For 3.0 sccm flow rate of O2, the formation of phases Ti3N2-x and Ti4N3-x are respectively noticed at 36.4 and 41.3°, which are in greater evidence than the other streams. The roughness parameters of untreated and plasma-treated samples are shown in Table 1. As can be clearly observed, plasma-treated surfaces exhibit significantly higher roughness values as compared with the untreated samples. The typical surface topography of the samples is shown in Fig. 2. According to Table 1, the roughness parameters showed no direct relationship with the flow rate of oxygen used in the plasma formation. The mechanisms of erosion and deposition generated by plasma discharge are acting simultaneously and result in an increased roughness as compared with the untreated sample. This is noted by the increase in the average maximum peaks and valleys, Rp and Rv (28). Rough surfaces of titanium are known to play a role in bacterial adhesion (29). Traditionally, a moderate roughness is used in titanium implants to promote
TABLE 1. Roughness parameter values and surface contact angle of titanium samples before and after plasma treatment Roughness parameters (nm) Samples (a) (b)
Untreated 1N2–4Ar–2O2 1N2–4Ar–3O2
Ra
Rz
Rp
Rv
Contact angle (θ)
0.43 ± 0.23 12.22 ± 1.07 16.54 ± 0.94
9.65 ± 0.49 249.86 ± 1.43 288.50 ± 1.72
7.82 ± 0.72 166.15 ± 2.79 189.82 ± 3.17
1.86 ± 0.57 83.71 ± 3.46 98.68 ± 4.61
66.34 ± 0.36 62.48 ± 0.21 75.17 ± 0.32
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FIG. 2. Nanotopography of titanium sample oxynitrided by plasma in atmosphere with different flow of oxygen (a) untreated, (b) 4Ar–1N2–2O2, (c) 4Ar–1N2–3O2. Typical topographic profiles for scanning areas of 15 μm × 15 μm.
osseointegration (30). Therefore, an ideal surface for a titanium implant would be one that promotes the adhesion of tissue cells and is not conducive to bacterial adhesion (6). The microtopography of the surface can directly influence the interaction between surface and bacteria (31). In our study, untreated surfaces were clearly more susceptible to bacterial adhesion when compared with the treated ones. However, the oxygen proportion seems to have a limit of interference on bacterial adhesion, as the treatment with 3.0 sccm of O2 presents a higher average roughness value (Table 1) but proved to be the less susceptible to bacterial adhesion as can be sharply seen in Fig. 3. Our results are in accordance with a previous work (32), which suggested that surfaces larger than bacterial colonies have insignificant
adhesion and retention. The untreated titanium sample (polished) has the smoothest surface (Ra = 0.43 μm), whereas the intermediate condition (3.0 sccm of O2) possesses the rougher surface (Ra = 16.54 μm) and the minimal S. epidermidis adhesion and retention. Similarly to the roughness test, wettability results showed that values of contact angle are not directly related to the oxygen flux. The value of the contact angle obtained for the untreated sample was 66.34°, which lies in between the values of the plasma-treated samples, as observed in Table 1. In this sense, it is possible to say that plasma treatment conditions used in this work can generate titanium surfaces with hydrophilicity higher or lower than untreated ones.
FIG. 3. Bacterial adhesion to Ti titanium disk surfaces at 6 and 24 h. (a) 4Ar–1N2–2O2; (b) 4Ar–1N2–3O2. Artif Organs, Vol. 2015 Vol. ••, 40, No. No. ••, 5, 2016
THOUGHTS AND PROGRESS 80
80
70 2
Surface tension (mJ/m )
p
75
d
70
γs
65
γs
60
γs= γs + γs
60
55
θ
55
65 p
d
50
50
45
45
40
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
Contact angle (θ)
75
0
Standard sample
2
3
Flow of O2 gas (sccm)
FIG. 4. Contact angle and surface tension for untreated titanium samples and those oxynitrided at different O2 flow rates.
Data from Ti surfaces wettability shows the hydrophilicity of the surfaces, as the contact angles were lower than 90°. Several authors have reported that hydrophilic materials are more resistant to bacterial adhesion than hydrophobic materials (3,33,34). However, we have noticed that although the untreated sample also had a low contact angle, there was bacterial growth and biofilm formation on its surface. Thus, the results of this work suggest that bacterial adhesion and subsequent proliferation are probably dependent on different surface properties, and are not limited to the influence of wettability. In all the different O2 flow rates used in the treatments, a competition between nitrogen and oxygen species present in the plasma occurs. This phenomenon promoted different behaviors with respect to the interaction of the droplet with the titanium surface. These changes in the hydrophobic and hydrophilic character of titanium are related to changes in polarity and dispersive components of surface tension (23). It is observed that for the flow rate of 3.0 sccm of O2, the value for the dispersive component together with surface tension value could have caused the formation of phases Ti3N2-x and Ti4N3-x (Fig. 4). Researchers have shown that bacterial adherence decreases with decreasing contact angle and increasing surface tension of synthetic materials (35). Scanning electron microscopy (SEM) pictures (Fig. 3) clearly shows that plasma-treated surfaces are less susceptible to bacterial adhesion. Interestingly, best results were obtained for the surface treated with 4Ar-1N2-3O2 condition (Fig. 3b). This phenomenon suggests the existence of an optimum O2 concentration for the plasma treatment, as lower oxygen concentrations (Fig. 3a; 4–Ar–1N2–
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2O2) exhibit more bacterial adhesion. This observation suggests that higher titanium oxidation benefits the biofilm dispersion. Important here is that either bacterial adhesion or biofilm dispersion occurs without the presence of a chemical stress (such as antibiotics) in all treatments performed. This is relevant to the clinical practice, because once the bacterium stays in the planktonic stage, they are more susceptible to antibiotics and to the host immune system as their attached counterparts (3). CONCLUSIONS The surface properties influence adherence, and therefore bacterial proliferation and retention. In this work, the samples that underwent plasma treatment presented lower biofilm formation. The flow rate of oxygen employed during plasma treatment plays a key role on the properties of the titanium surfaces. An ideal proportion of the gases used was achieved to minimize S. epidermidis adhesion, which is the most important biomedical infections-related microorganism. It is important to notice that early stages of biofilm growth were inhibited and this result shows the potential application of this treatment on titanium surfaces with anti-infectious biomedical interest. REFERENCES 1. Chandra J, Patel JD, Li J, et al. Modification of surface properties of biomaterials influences the ability of Candida albicans to form biofilms. Appl Environ Microbiol 2005;71:8795–801. 2. Karunakaran E, Mukherjee J, Ramalingam B, Biggs CA. “Biofilmology”: a multidisciplinary review of the study of microbial biofilms. Appl Microbiol Biotechnol 2011;90:1869– 81. 3. Treter J, MacEdo AJ. Science against microbial pathogens: communicating current research and technological advances. cap. 2. In: Mendez-Vilas A, ed. Science against Microbial Pathogens: Communicating Current Research and Technological Advances, vol. 2. Badajoz, Spain: Formatex Research Center, 2011;693–1348. 4. Liu X, Chu PK, Ding C. Surface modification of titanium, titanium alloys, and related materials for biomedical applications. Mater Sci Eng Rep 2004;47:49–121. 5. Truong VK, et al. The influence of nano-scale surface roughness on bacterial adhesion to ultrafine-grained titanium. Biomaterials 2010;31:3674–83. 6. Wu Y, et al. Differential response of Staphylococci and osteoblasts to varying titanium surface roughness. Biomaterials 2011;32:951–60. 7. Rino J-P, Studart N. Structural correlations in titanium dioxide. Phys Rev B 1999;59:6643–9. 8. Lu X, Leng Y. TEM study of calcium phosphate precipitation on bioactive titanium surfaces. Biomaterials 2004;25:1779–86. 9. Shirkhanzadeh M. Fabrication and characterization of alkoxy-derived nanophase TiO2 coatings. Nanostruct Mater 1995;5:33–40. 10. Windecker S, Simon R, Lins M, et al. Randomized comparison of a titanium-nitride-oxide-coated stent with a stainless steel stent for coronary revascularization: the TiNOX trial. Circulation 2005;111:2617–22. Artif ArtifOrgans, Organs,Vol. Vol.••, 40,No. No.••, 5, 2015 2016
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11. Koerner RJ, et al. Bacterial adhesion to titanium-oxynitride (TiNOX) coatings with different resistivities: a novel approach for the development of biomaterials. Biomaterials 2002;23:2835–40. 12. Poortinga AT, Bos R, Busscher HJ. Charge transfer during staphylococcal adhesion to TiNOX® coatings with different specific resistivity. Biophys Chem 2001;91:273–9. 13. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the Natural environment to infectious diseases. Nat Rev Microbiol 2004;2:95–108. 14. David D. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2003;2:114–22. 15. Bridgett MJ, Davies MC, Denyer SP. Control of staphylococcal adhesion to polystyrene surfaces by polymer surface modification with surfactants. Biomaterials 1992;13:411–6. 16. Michael O. Staphylococcus epidermidis—the “accidental” pathogen. Nat Rev Microbiol 2009;7:555–67. 17. Zhao L, et al. Antibacterial coatings on titanium implants. J Biomed Mater Res B Appl Biomater 2009;91B:470–80. 18. Antoci V Jr, Adams CS, Parvizi J, et al. The inhibition of Staphylococcus epidermidis biofilm formation by vancomycinmodified titanium alloy and implications for the treatment of periprosthetic infection. Biomaterials 2008;29:4684–90. 19. Kim W-H, et al. The release behavior of CHX from polymercoated titanium surfaces. Surf Interf Anal 2008;40:202–4. 20. Gosheger G, et al. Silver-coated megaendoprostheses in a rabbit model—an analysis of the infection rate and toxicological side effects. Biomaterials 2004;25:5547–56. 21. Sadiqali C, et al. Gas plasmas and plasma modified materials in medicine. J Appl Biomed 2010;8:55–66. 22. Puckett SD, et al. The relationship between the nanostructure of titanium surfaces and bacterial attachment. Biomaterials 2010;31:706–13. 23. Costa THC, et al. Caracterização de filmes de poliéster modificados por plasma de O2 a baixa pressão. Matéria 2008;13:65–76.
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24. Sá JC, et al. Influence of argon-ion bombardment of titanium surfaces on the cell behavior. Surf Coat Technol 2009;203:1765–70. 25. Öner D, McCarthy TJ. Ultrahydrophobic surfaces. Effects of topography length scales on wettability. Langmuir 2000;16:7777–82. 26. Deshmukh R, Bhat N. The mechanism of adhesion and printability of plasma processed PET films. Mater Res Innov 2003;7:283–90. 27. Trentin DDS, et al. Potential of medicinal plants from the Brazilian semi-arid region (Caatinga) against Staphylococcus epidermidis planktonic and biofilm lifestyles. J Ethnopharmacol 2011;137:327–35. 28. Whitehead SA, et al. Comparison of methods for measuring surface roughness of ceramic. J Oral Rehabil 1995;22:421–7. 29. Quirynen M Fau, Bollen CM, et al. The influence of titanium abutment surface roughness on plaque accumulation and gingivitis: short-term observations. Int J Oral Maxillofac Implants 1996;11:169–78. 30. Wall I, Donos N, Carlqvist K, Jones F, Brett P. Modified titanium surfaces promote accelerated osteogenic differentiation of mesenchymal stromal cells in vitro. Bone 2009;45:17– 26. 31. Araújo EA, et al. Aspectos coloidais da adesão de microorganismos. Química Nova 2010;v:1940–8. 32. Whitehead KA, Verran J. The effect of surface topography on the retention of microorganisms. Food Biop Proc 2006;84:253–9. 33. Darouiche RO. Device-associated infections: a macroproblem that starts with microadherence. Clin Infect Dis 2001;33:1567– 72. 34. Pascual A. Pathogenesis of catheter-related infections: lessons for new designs. Clin Microbiol Infect 2002;8:256–64. 35. Locatelli CI, et al. Aderência bacteriana in vitro a lentes intraoculares de polimetilmetacrilato e de silicone. Arq Bras Oftalmol 2004;67:241–8.
Copyright V © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc. Copyright C 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Thoughts and Progress Influence of Oxynitrided Surface in the Production of a Less Susceptible Titanium Surface to Skin-Borne Bacterial Adhesion *Michelle de Medeiros Aires, †Janine Treter, *Danilo Cavalcante Braz, ‡Cristiano Krug, †Alexandre José Macedo, and *Clodomiro Alves Júnior *Lab Plasma, Federal University of Rio Grande do Norte, Rio Grande do Norte; †Biofilms and Microbial Diversity Laboratory, Biotechnology Center and Pharmacy College of the Federal University of Rio Grande do Sul; and ‡Physical Institute, Federal University of Rio Grande do Sul, Rio Grande do Sul, Brazil Abstract: There is a growing quest for an ideal biomaterial that shows appropriate cellular response and is not susceptible to microbial adhesion. In this study, commercial grade II titanium was submitted to RF/DC plasma surface modification at 2.2 mbar, using gas mixtures of argon, nitrogen, and oxygen at proportions 4:1:2 and 4:1:3. The surfaces were physically and chemically characterized. In order to evaluate bacterial response, the surfaces were exposed to Staphylococcus epidermidis. Oxynitrided samples, although having a higher roughness as compared with untreated samples, exhibited lower bacterial growth. This observation is probably due to the formation of different crystalline phases of nitrides and oxides caused by plasma treatment. The surface with highest contact angle and highest surface tension showed lower bacterial adhesion. These results were confirmed by scanning electron microscopy. The role of nitrogen in reducing bacterial adhesion is clear when this material is compared with untreated titanium, on which only an oxide film is present. Key Words: Plasma— Staphylococcus epidermidis—Biofilm—Titanium.
The increasing demand for implants and medical devices has led to a concomitant increase in the incidence of device-related infections (1). It is estimated that approximately 65% of hospital-acquired infections are related to surface-attached microorganisms (2). When any biomaterial is implanted, it can doi:10.1111/aor.12581 Received February 2015; revised June 2015. Address correspondence and reprint requests to Miss Michelle de Medeiros Aires, LabPlasma, Universidade Federal do Rio Grande do Norte, 59072-970 Rio Grande do Norte, Brazil. E-mail: [email protected]
become a site for bacterial adhesion, colonization, and infection (3). Bacteria tend to adhere to different types of surfaces, ranging from surfaces in the human body, and plants and clays, to plastic and metals (4). Among the metals, titanium and its alloys have increased their use as biomaterial stems, mainly in orthopedic and dental implants (5) due to their mechanical strength, biocompatibility, and corrosion resistance (6). The chemical and physical stability of titanium dioxide, TiO2, led to an increased attention toward this titanium compound in the last decade (7). The surface energy, thickness, roughness, and crystalline properties of this material are widely studied (8). It is well established in literature that titanium biocompatibility is connected with a nano-TiO2 layer on its surface (9). Intermediate stoichiometric titanium compounds (TiNxOy) began to be studied due to the ability of the nitrogen to act as a dopant in TiO2. The properties of the compound are strongly dependent on the ratio oxygen/nitrogen (10). Characteristics such as changing gap, resistivity, surface electron acceptors, or donors affect bacterial adherence on the surface. Staphylococcus epidermidis adheres minimally to the TiNxOy films deposited by physical vapor deposition with specific values of resistivity (11). It was found that the load transfer between TiNxOy surfaces and bacteria during the adhesion process depends upon the substrate specific resistivity and provides a strong influence on bacterial adhesion (12). Once the bacteria are adhered to the medical device, they start a multifactor process of biofilm formation. Biofilms are a protected mode of life of bacteria, where they are adhered on a surface embedded in a self-produced matrix (13), becoming more resistant to antibiotics and the host immune system (14). Most biomaterial-related infections are caused by skin commensals, in particular the coagulasenegative staphylococci (15) like S. epidermidis (16). To minimize microbial adhesion on titanium surfaces, several approaches have been made in recent years. However, antibacterial coatings are a complex issue, as they should be tailored to specific bacterial species (17). Some examples of attempts include antibiotic-loaded coatings (18), organic bactericide coatings (19), and inorganic bactericide surfaces like silver coating (20). ArtificialOrgans Organs2016, 2015,40(5):521–526 ••(••):••–•• Artificial
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THOUGHTS AND PROGRESS
It is well known that initial steps of biofilm formation are closely related to bacterial adhesion on the surface of the material, which is crucial for the success of bacterial colonization. Therefore, new strategies focus on changing physicochemical properties of the surfaces. These methods are relatively simple and an economic way to repel bacteria colonization (17). Among the different surface modification methods available, those that use plasma as a source of energy advanced considerably in recent years, mainly due to their versatility (6,21). Moreover, bacterial adhesion reduction on medical devices by a process without any drug incorporation is an important strategy to avoid nosocomial infections (22). In this work, modification of titanium surface by plasma was evaluated as a means of avoiding adhesion of the pathogenic skin-borne bacteria— S. epidermidis. The surface modification procedure was performed by using different gas compositions, where the influence of that parameter was carefully studied. MATERIALS AND METHODS Material and plasma treatment Titanium samples were prepared from Ti cp (Degree II). They were cut in a 9 mm × 3 mm (diameter × thickness) disk format. Initially, the titanium disks were sanded with silicon carbide sandpaper and polished by a porous neoprene (PANTEC, Zwick Roell AG, Ulm, Germany) impregnated with colloidal silica and hydrogen peroxide mixture solution for a 30-min period. The samples were afterwards washed with enzymatic detergent followed by absolute ethanol and distilled water. The samples were kept under ultrasonic agitation for 10 min at every step (23). Polished titanium disks without plasma treatment were used as reference in each assay. Surface modification of titanium disks was carried out in a cylindrical (300 mm × 300 mm) plasma reactor made of stainless steel, equipped with a direct current power supply and a vacuum system as previously described (24). Before the treatment of the disks was carried out, a precleaning step was performed in order to remove oxides and other contaminants using ionic bombardment. For this step, the following parameters were used: 2.0 sccm argon and 2.0 sccm hydrogen flow rates, 1.5 mbar pressure, 200°C temperature, and 594 V voltage. These conditions allowed formation of plasma, which contained neutral species as well as charged ones, such as Ar1+, H1+, and H2+. These species bombed and pulled out material at the surface of the sample. This sputtering and chemical erosion step was maintained for 30 min. Artif Organs, Vol. 2015 Vol. ••, 40, No. No. ••, 5, 2016
For plasma treatment itself, three different atmosphere conditions were used, where only the flow of oxygen was varied. The conditions employed were 4.0 sccm Ar, 1.0 sccm N2, and 2.0 or 3.0 sccm O2.The working pressure was set at 2.2 mbar and temperature during treatment was kept constant at 500°C for 1 h. Afterwards, the equipment was turned off and the samples were allowed to cool down in the reactor chamber. Surface characterization X-ray diffraction (XDR) measurements were carried out at grazing angle of 1° using the Shimadzu6000 diffractometer (Shimadzu Corporation, Kyoto, Japan), Cu Kα radiation at a 2θ scan from 25.0 to 45.0°. The surface topography and roughness of untreated and plasma-treated disks were measured with an atomic force microscope (AFM) (SPM9600, Shimadzu). Four roughness parameters were evaluated: average roughness (Ra), average maximum height of the profile (Rz), maximum profile peak height (Rp), and maximum profile valley depth (Rv). Nanotopography images were taken in three different regions of the sample. In each 2D image, lines were obtained for determination of roughness profiles. Surface wettability measurements were also conducted. A precleaning procedure using 0.5% of enzymatic detergent in double-distilled water followed by an ultrasonic treatment for 10 min was performed. The disks (untreated and plasma treated) were washed with absolute ethanol and subsequently with distilled water. The sessile drop method was employed to measure the static contact angles of different solvents on titanium disks (25). Doubledistilled water and glycerol (Vetec Quimica, Rio de Janeiro, Brazil) were used as previously described (24). The contact angle measurements were executed by dripping 20 μL of the solvent on the sample surface using a custom-made device (23). An average value of the contact angle was obtained from at least six measurements for each solvent/titanium surface combination. Images were taken at different times until complete relaxation of the drop with Pinnacle Studio 9.0 program (Corel Corporation, Ottawa, Canada). The measurement of surface tension was based on the drop geometric method developed by Fowkes (26). Microorganisms, culture conditions, and sample preparation Staphylococcus epidermidis ATCC 35984 cells were grown aerobically at 37°C for 24 h on MuellerHinton agar plates (OXOID) from frozen stocks. Several colonies were used to make the bacterial
THOUGHTS AND PROGRESS
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FIG. 1. X-Ray diffractogram in GIXRD of titanium disk samples: (a) untreated and (b) 4Ar–1N2–2O2 in solid line and 4Ar–1N2– 3O2 in dotted line.
suspension in sterile sodium chloride 0.9% at a final concentration of 3 × 108 CFU per mL. Biofilm formation method (27) was adapted to 24-well plates containing a sterile titanium disk. Briefly, in each well 400 μL of the bacterial suspension and 600 μL of tryptone soy broth were added (TSB) (OXOID). After incubation period of 6 and 24 h at 37°C, the titanium samples were washed twice with sterile 0.9% sodium chloride to remove free bacterial cells and fixed for 2 h in glutaraldehyde 2.5%. Then the samples were washed with 100 mM of sodium cacodylate buffer (pH 7.2) and treated for 2 h with 2% osmium tetroxide. After that, the samples were again washed with 100 mM of sodium cacodylate buffer (pH 7.2) and finally dehydrated using increasing concentrations of acetone. The dehydrated titanium disks were dried by the CO2 critical point technique (Balzers CPD 030, Leica Microsystems GmbH, Wetzlar, Germany), fixed on aluminum stubs, covered with a gold film, and examined in a JEOL JSM-6060 scanning electron microscope (JEOL USA, Inc., Peabody, MA, USA). RESULTS AND DISCUSSION Figure 1 shows the XRD analysis of untreated and treated samples with different O2 flow rates. The
results demonstrate the formation of different crystalline phases of oxides and nitrides on the titanium surface, such as TiO6, TiO2, Ti3N2x, and Ti4N3-x. The presence of TiO2 phase is observed at 27°, where the peak intensity increases with increasing oxygen gas flux in the treatments. For 3.0 sccm flow rate of O2, the formation of phases Ti3N2-x and Ti4N3-x are respectively noticed at 36.4 and 41.3°, which are in greater evidence than the other streams. The roughness parameters of untreated and plasma-treated samples are shown in Table 1. As can be clearly observed, plasma-treated surfaces exhibit significantly higher roughness values as compared with the untreated samples. The typical surface topography of the samples is shown in Fig. 2. According to Table 1, the roughness parameters showed no direct relationship with the flow rate of oxygen used in the plasma formation. The mechanisms of erosion and deposition generated by plasma discharge are acting simultaneously and result in an increased roughness as compared with the untreated sample. This is noted by the increase in the average maximum peaks and valleys, Rp and Rv (28). Rough surfaces of titanium are known to play a role in bacterial adhesion (29). Traditionally, a moderate roughness is used in titanium implants to promote
TABLE 1. Roughness parameter values and surface contact angle of titanium samples before and after plasma treatment Roughness parameters (nm) Samples (a) (b)
Untreated 1N2–4Ar–2O2 1N2–4Ar–3O2
Ra
Rz
Rp
Rv
Contact angle (θ)
0.43 ± 0.23 12.22 ± 1.07 16.54 ± 0.94
9.65 ± 0.49 249.86 ± 1.43 288.50 ± 1.72
7.82 ± 0.72 166.15 ± 2.79 189.82 ± 3.17
1.86 ± 0.57 83.71 ± 3.46 98.68 ± 4.61
66.34 ± 0.36 62.48 ± 0.21 75.17 ± 0.32
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FIG. 2. Nanotopography of titanium sample oxynitrided by plasma in atmosphere with different flow of oxygen (a) untreated, (b) 4Ar–1N2–2O2, (c) 4Ar–1N2–3O2. Typical topographic profiles for scanning areas of 15 μm × 15 μm.
osseointegration (30). Therefore, an ideal surface for a titanium implant would be one that promotes the adhesion of tissue cells and is not conducive to bacterial adhesion (6). The microtopography of the surface can directly influence the interaction between surface and bacteria (31). In our study, untreated surfaces were clearly more susceptible to bacterial adhesion when compared with the treated ones. However, the oxygen proportion seems to have a limit of interference on bacterial adhesion, as the treatment with 3.0 sccm of O2 presents a higher average roughness value (Table 1) but proved to be the less susceptible to bacterial adhesion as can be sharply seen in Fig. 3. Our results are in accordance with a previous work (32), which suggested that surfaces larger than bacterial colonies have insignificant
adhesion and retention. The untreated titanium sample (polished) has the smoothest surface (Ra = 0.43 μm), whereas the intermediate condition (3.0 sccm of O2) possesses the rougher surface (Ra = 16.54 μm) and the minimal S. epidermidis adhesion and retention. Similarly to the roughness test, wettability results showed that values of contact angle are not directly related to the oxygen flux. The value of the contact angle obtained for the untreated sample was 66.34°, which lies in between the values of the plasma-treated samples, as observed in Table 1. In this sense, it is possible to say that plasma treatment conditions used in this work can generate titanium surfaces with hydrophilicity higher or lower than untreated ones.
FIG. 3. Bacterial adhesion to Ti titanium disk surfaces at 6 and 24 h. (a) 4Ar–1N2–2O2; (b) 4Ar–1N2–3O2. Artif Organs, Vol. 2015 Vol. ••, 40, No. No. ••, 5, 2016
THOUGHTS AND PROGRESS 80
80
70 2
Surface tension (mJ/m )
p
75
d
70
γs
65
γs
60
γs= γs + γs
60
55
θ
55
65 p
d
50
50
45
45
40
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
Contact angle (θ)
75
0
Standard sample
2
3
Flow of O2 gas (sccm)
FIG. 4. Contact angle and surface tension for untreated titanium samples and those oxynitrided at different O2 flow rates.
Data from Ti surfaces wettability shows the hydrophilicity of the surfaces, as the contact angles were lower than 90°. Several authors have reported that hydrophilic materials are more resistant to bacterial adhesion than hydrophobic materials (3,33,34). However, we have noticed that although the untreated sample also had a low contact angle, there was bacterial growth and biofilm formation on its surface. Thus, the results of this work suggest that bacterial adhesion and subsequent proliferation are probably dependent on different surface properties, and are not limited to the influence of wettability. In all the different O2 flow rates used in the treatments, a competition between nitrogen and oxygen species present in the plasma occurs. This phenomenon promoted different behaviors with respect to the interaction of the droplet with the titanium surface. These changes in the hydrophobic and hydrophilic character of titanium are related to changes in polarity and dispersive components of surface tension (23). It is observed that for the flow rate of 3.0 sccm of O2, the value for the dispersive component together with surface tension value could have caused the formation of phases Ti3N2-x and Ti4N3-x (Fig. 4). Researchers have shown that bacterial adherence decreases with decreasing contact angle and increasing surface tension of synthetic materials (35). Scanning electron microscopy (SEM) pictures (Fig. 3) clearly shows that plasma-treated surfaces are less susceptible to bacterial adhesion. Interestingly, best results were obtained for the surface treated with 4Ar-1N2-3O2 condition (Fig. 3b). This phenomenon suggests the existence of an optimum O2 concentration for the plasma treatment, as lower oxygen concentrations (Fig. 3a; 4–Ar–1N2–
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2O2) exhibit more bacterial adhesion. This observation suggests that higher titanium oxidation benefits the biofilm dispersion. Important here is that either bacterial adhesion or biofilm dispersion occurs without the presence of a chemical stress (such as antibiotics) in all treatments performed. This is relevant to the clinical practice, because once the bacterium stays in the planktonic stage, they are more susceptible to antibiotics and to the host immune system as their attached counterparts (3). CONCLUSIONS The surface properties influence adherence, and therefore bacterial proliferation and retention. In this work, the samples that underwent plasma treatment presented lower biofilm formation. The flow rate of oxygen employed during plasma treatment plays a key role on the properties of the titanium surfaces. An ideal proportion of the gases used was achieved to minimize S. epidermidis adhesion, which is the most important biomedical infections-related microorganism. It is important to notice that early stages of biofilm growth were inhibited and this result shows the potential application of this treatment on titanium surfaces with anti-infectious biomedical interest. REFERENCES 1. Chandra J, Patel JD, Li J, et al. Modification of surface properties of biomaterials influences the ability of Candida albicans to form biofilms. Appl Environ Microbiol 2005;71:8795–801. 2. Karunakaran E, Mukherjee J, Ramalingam B, Biggs CA. “Biofilmology”: a multidisciplinary review of the study of microbial biofilms. Appl Microbiol Biotechnol 2011;90:1869– 81. 3. Treter J, MacEdo AJ. Science against microbial pathogens: communicating current research and technological advances. cap. 2. In: Mendez-Vilas A, ed. Science against Microbial Pathogens: Communicating Current Research and Technological Advances, vol. 2. Badajoz, Spain: Formatex Research Center, 2011;693–1348. 4. Liu X, Chu PK, Ding C. Surface modification of titanium, titanium alloys, and related materials for biomedical applications. Mater Sci Eng Rep 2004;47:49–121. 5. Truong VK, et al. The influence of nano-scale surface roughness on bacterial adhesion to ultrafine-grained titanium. Biomaterials 2010;31:3674–83. 6. Wu Y, et al. Differential response of Staphylococci and osteoblasts to varying titanium surface roughness. Biomaterials 2011;32:951–60. 7. Rino J-P, Studart N. Structural correlations in titanium dioxide. Phys Rev B 1999;59:6643–9. 8. Lu X, Leng Y. TEM study of calcium phosphate precipitation on bioactive titanium surfaces. Biomaterials 2004;25:1779–86. 9. Shirkhanzadeh M. Fabrication and characterization of alkoxy-derived nanophase TiO2 coatings. Nanostruct Mater 1995;5:33–40. 10. Windecker S, Simon R, Lins M, et al. Randomized comparison of a titanium-nitride-oxide-coated stent with a stainless steel stent for coronary revascularization: the TiNOX trial. Circulation 2005;111:2617–22. Artif ArtifOrgans, Organs,Vol. Vol.••, 40,No. No.••, 5, 2015 2016
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