Inhibitory effect of super-hydrophobicity on silver release and antibacterial properties of super-hydrophobic Ag/TiO2 nanotubes Licheng Zhang,1* Lihai Zhang,1* Yun Yang,2* Wei Zhang,1 Houchen Lv,1 Fei Yang,3 Changjian Lin,2 Peifu Tang1 1
Department of Orthopaedics, General Hospital of Chinese PLA, Beijing 100853, China State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China 3 Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China 2
Received 7 November 2014; revised 28 April 2015; accepted 2 May 2015 Published online 20 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33454 Abstract: The antibacterial properties of super-hydrophobic silver (Ag) on implant surface have not yet to be fully illuminated. In our study, we investigate the protective effects of super-hydrophobic coating of silver/titanium dioxide (Ag/TiO2) nanotubes against bacterial pathogens, as well as its pattern of Ag release. Ag/TiO2 nanotubes are prepared by a combination of electrochemical anodization and pulse electrodeposition. The super-hydrophobic coating is prepared by modifying the surface of Ag/TiO2 nanotubes with 1H, 1H, 2H, 2Hperfluorooctyl-triethoxysilane (PTES). Surface features and Ag release are examined by SEM, X-ray photoelectron spectroscopy, contact-angle measurement, and inductively coupled plasma-mass spectrometry (ICP-MS). The antibacterial activity of super-hydrophobic coating Ag/TiO2 nanotubes is investigated both in vitro and in vivo. Consequently, the superhydrophobic coating on Ag/TiO2 nanotubes shows a regularly
arranged structure; and nano-Ag particles (10–30 nm) are evenly distributed on the surface or inside the nanotubes. The contact angles of water on the super-hydrophobic coating Ag/ TiO2 nanotubes are all above 1508. In addition, the superhydrophobic character displays a certain conserved effect that contributes to the sustained release of Ag. The superhydrophobic Ag/TiO2 nanotubes are also effective in inhibiting bacterial adhesion, killing the adhering bacteria and preventing postoperative infection in rabbits. Therefore, it is expected that the super-hydrophobic Ag/TiO2 nanotubes which can contain the release of Ag, leading to stable release, may show a conC 2015 Wiley Periodicals, sistent surface antibacterial capability. V Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1004–1012, 2016.
Key Words: Ag/TiO2 nanotubes, super-hydrophobic, silver release, bacterial adhesion, antibacterial activity
How to cite this article: Zhang L, Zhang L, Yang Y, Zhang W, Lv H, Yang F, Lin C, Tang P. 2016. Inhibitory effect of superhydrophobicity on silver release and antibacterial properties of super-hydrophobic Ag/TiO2 nanotubes. J Biomed Mater Res Part B 2016:104B:1004–1012.
INTRODUCTION
Since the 1990s, when Barthlott et al.1 clarify the selfcleaning effect of the surface of lotus leaves and the superhydrophobic phenomenon, super-hydrophobic surfaces have attracted wide attention.2–4 The super-hydrophobicity of lotus leaves results from the micro-nano structure of their surfaces: the surface of every micrometer mastoid is attached to numerous nanoscale wax crystal particles. This micro-nano structure can significantly increase the contact angle of water droplets on the surface so that they easily fall off. Hydrophilcity or hydrophobicity of a surface affects protein and bacterial adhesion.5 A hydrophilic surface (contact angle < 908) can accelerate the adhesion of bacteria such as
Staphylococcus aureus and Escherichia coli; however, a hydrophobic surface (contact angle 90–1508) can inhibit the adhesion of Deinococcus geothermalis and Meiothermus silvanus6; as well, S. aureus and Streptococcus mutans cannot easily adhere to the surface of hydrophobic materials.7,8 Thus, bacterial adhesion can be inhibited and infection risk reduced with super-hydrophobic coated materials. Only a few reports have shown the inhibitory effect of super-hydrophobic coating on bacterial adhesion.9–12 Poncin-Epaillard et al.11 fabricate a highly hydrophobic polymeric surface that can reduce the adhesion of both Gram positive and negative pathogenic bacteria. Schoenfisch et al.9 synthesize a super-hydrophobic gel coating that can
Additional Supporting Information may be found in the online version of this article. *These authors contributed equally to this work. Correspondence to: P. Tang; e-mail: [email protected] or C. Lin; e-mail: [email protected] Contract grant sponsor: The National Natural Science Foundation of China; contract grant number: 81171690 Contract grant sponsor: PLA Postgraduate Medical School; contract grant number: 11BCZ02
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significantly inhibit adhesion by S. aureus and Pseudomonas aeruginosa. Crick et al.10 prepare an elastic superhydrophobic coating that can inhibit the adhesion of E. coli and methicillin-resistant S. aureus. The super-hydrophobic surface can inhibit bacterial adhesion but does not kill bacteria; whether an antibacterial substance can be incorporated into a super-hydrophobic surface is unknown. Silver (Ag) has been widely used in every field of biomedicine because of its broad antibacterial spectrum, excellent thermal stability and desirable biological safety.13 Ag has powerful antibacterial properties and the lowest toxicity among the metal antibacterial agents.14 Therefore, introducing Ag with its enhanced antibacterial properties into a super-hydrophobic surface can be used to produce a superior antibacterial super-hydrophobic surface. Superhydrophobic Ag cotton fabrics successfully developed by Yazdanshenas et al.15 can effectively inhibit bacterial adhesion, and the Ag ion on the surface can kill a few E. coli and S. aureus bacteria adhering to and around the surface, for excellent antibacterial performance. However, no study has investigated the antibacterial effects of a super-hydrophobic Ag surface in biomedicine. As well, air captured by the super-hydrophobic surface can reduce the contact area between the material and body fluid as well as slow the speed of drug release,16,17 which can provide long-term release of Ag. In this study, we create Ag-supported/TiO2 nanotubes by a combination of electrochemical anodization and pulse electrodeposition and prepare a super-hydrophobic coating by modifying the surface of the Ag/TiO2 nanotubes with 1H, 1H, 2H, 2H-perfluorooctyl-triethoxysilane (PTES). We evaluate the inhibitory effect of the coating on bacterial adhesion and killing adherent bacteria and assess its effect on Ag release. METHODS
Preparation of coated Ag/TiO2 nanotubes Titanium (Ti) foil (99.6% pure Ti) is cut into 10 3 10 3 0.1 mm3. Titanium rods are cut into cylindrical geometry of 30 mm in length and 2.5 mm in diameter. Samples are polished by sandpaper, cleaned by ultrasonic vibration, then TiO2 nanotubes were fabricated in 0.5 wt % HF (Hydrofluoric acid) electrolyte using the previously reported electrochemical anodizing technique.18,19 Titanium sample was connected to the power supply as anode, and platinum plate electrode was selected as the cathode. Prepared samples are cleaned by ultrasonic vibration and placed after drying in a muffle furnace at constant temperature of 4508C for 2 h, to transform the amorphous TiO2 into anatase. Pulse electrodeposition is used to deposit Ag in the formed TiO2 nanotubes.20 At room temperature, nano-Ag particles deposition was carried out in an aqueous solution of 10 mM AgNO3 (silver nitrate) and 100 mM NaNO3 (sodium nitrate). The TiO2 nanotube sample and Pt plate were used as the working and counter electrodes, respectively. Nano-Ag particles are synthesized in a electrochemical workstation (Autolab PGSTAT 30, Eco Echemine BV Company, The Netherlands) with current density 10 mA cm22 and an alternating
procedure of opening every 0.1 s and closing every 0.3 s as well as deposition time 300 circles (cycle) to produce Ag/ TiO2 nanotubes (Ag/TNT). Samples are cleaned by ultrasonic vibration and preserved in a vacuum after drying. Test of physicochemical characteristics of coated Ag/ TiO2 nanotubes Field Emission Scanning Electron Microscope (Hitachi S4800, Hitachi High-Technologies Corporation, Japan) was employed to characterize the surface morphology of the Ag/ TiO2 Nanotubes. The Ag concentration of the surface of Ag/ TiO2 Nanotubes was determined by energy-dispersive X-ray spectroscopy (Hitachi S-4800, Hitachi High-Technologies Corporation, Japan). X-ray photoelectron spectrometer was utilized to determine the Ag distribution in the Ag/TiO2 nanotubes (VGMultiLab 2000, Thermal Electron, USA), with Al Ka as the radiation source and C1s 5 284.8 eV as the base of binding energy. Preparation of the super-hydrophobic coating of Ag/ TiO2 nanotubes Samples (TNT, Ag/TNT) are soaked in methanol solution with 1% 1H, 1H, 2H, 2H-perfluorooctyl-triethoxysilane (PTES; Degussa) for 1 h, and then placed in a drying oven at 1408C for 1 h. The PTES-modified sample is recorded as super-hydrophobic TiO2 nanotubes (S-TNT) and superhydrophobic Ag/TiO2 nanotubes (S-Ag/TNT). Samples are disinfected by irradiation with cobalt-60, and then the surface wettability was evaluated by measuring the contact angle between the sample and water droplets. Contact angle measurements were performed at room temperature using a Contact angle system (OCA 20, Data Physics, Germany). Effect of super-hydrophobic property on Ag release The amount of Ag released from the Ag/TNT or S-Ag/TNT samples was monitored in phosphate buffered saline (0.01 M PBS) at 378C. The samples were immersed in 6 mL of PBS for 24 h in the thermostated container, taken out, and then immersed again in 6 mL of fresh PBS. This process is repeated for 14 days. The concentration of Ag released is measured by ICP-MS (ELAN 6100, Perkin-Elmer, Waltham, MA). In vitro antimicrobial test of Ag/TNT and S-Ag/TNT Samples (TNT, S-TNT, Ag/TNT, S-Ag/TNT) are placed in sterile 24-well plates, and 2 mL S. aureus 8325 solution (1 3 106 colony forming units [cfu]/mL) is placed onto the surface of each sample. Plates are placed in an incubator with relative humidity > 90% and temperature 378C. After 2 or 4 h, plates are removed and washed with sterile PBS to remove non-adhering bacteria. Some samples are stained with LIVE/DEAD fluorescence staining reagent (Molecular Probes). After samples are stained away from the light for 15 min, they are washed with PBS. The distribution of bacteria was observed under a fluorescent microscope, and bacteria with green fluorescent (live bacteria) were counted in a field 200 3 200 mm2 by use of Image-Pro (MediaCybernetics, USA). Six views of TNT, S-TNT, Ag/TNT, S-Ag/TNT were selected for counting. Samples are fixed with 2.5%
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glutaral solution, kept in a 48C refrigerator overnight, dehydrated and critical point drying, and then spraying the surface of the sample thin gold film. At last, the samples are observed by SEM. In vivo antimicrobial test of S-Ag/TNT S-Ag/TNT implantation. We use 15 healthy male New Zealand white rabbits aged 8 months (body weight 2.5–3 kg) from the Laboratory Animal Center of PLA General Hospital. Total 30 hind legs are randomly divided into three groups (n 5 10 each): Titanium rods of TNT structured surfaces (TNT Group), Titanium rods of Ag/TNT structured surfaces (Ag/TNT Group) and Titanium rods of S-Ag/TNT structured surfaces (S-Ag/TNT Group). The animals are anesthetized with a 0.3 mL kg21 mixture of ketamine and sumianxin via intramuscular injection, areas are disinfected, and a sterile towel is applied. A vertical incision parallel to the femur is made at the lower end of femur and lateral knee joint, and skin, subcutaneous tissue and medial patellar ligament are cut away layer by layer, before the articular cavity is opened to disclose the femoral condyle. At 0.5 cm over the cross point of the femoral axis and the connection line of bilateral femoral condyles, bone tissue is reamed toward the proximal femur successively with use of Kirschner wire and different types of drill bits, before the medullary cavity is flushed with normal saline. An amount of 1 3 104 cfu mL21 S. aureus liquid (20 lL) is injected into the medullary cavity, before the TNT samples are implanted into the medullary cavity of distal femur, and the opening at the femoral condyle is closed with use of bone wax. The articular cavity is flushed with normal saline, and closed, before the incision is sutured layer by layer. After surgery, no antibiotic treatment is applied, and the incision is wiped with ethanol every day during postoperative 1 week. Postoperative radiography. Radionuclide bone scanning of bilateral femurs is conducted at postoperative weeks 1 and 2 for 2 to 4 h after injection of 99Tc-MDP, 10 mG, with acquisition interval (upper collection distance) 3 cm. The region of interest is at the symmetrical area of bilateral lower femurs. Radiographs are taken at postoperative weeks 2 and 4. Bacterial culture of bacteria eluent from implants. After animals are anesthetized by an overdose of ketamine and sumianxin and sacrificed, bilateral femurs are excised under aseptic conditions. At 3 cm from the screw entrance point at the femoral condyle, femurs are sawed off transversely to take out the nanotube. The surface of the nanotube is washed with normal saline solution to remove excess blood or bacteria, before cleaned by ultrasonography in 2 mL normal saline to remove adhered bacteria. Bacterial eluent removed is used for culture. Histology. Distal femoral bone specimens are fixed with 10% neutral formalin for 3 days, decalcification with 10% EDTA, embedded in paraffin and sectioned, before undergo hematoxylin and eosin (H&E) and Masson trichrome staining.
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Statistical analysis Data are described with mean 6 SD. Differences between groups are compared by one-way ANOVA and Student–Newman–Keuls test. Analysis involves use of StatView (SAS, Cary, NC). p < 0.05 is considered statistically significant. RESULTS
SEM and EDS of Ag/TiO2 nanotubes Pulse-electrodeposited samples are analyzed by SEM and EDS [Figure 1(D)]. The EDS spectral peaks (Working conditions: Acc.V 15.0 KV, mag400) reveal 3 elements: Ti, O, and Ag. After Ag is introduced onto the surface of samples by pulse electrodeposition, the shape of the tube does not change as compared with the pure TiO2 nanotube [Figure 1(A) and supplymentary Figure 1, Working conditions: Acc.V 20.0 KV, WD 10.5 mm, mag100 K]; however, oval or round particles of 10–30 nm are regularly distributed on the surface and at the orifice of TiO2 nanotubes [Figure 1(B), Working conditions: Acc.V 15.0 kV, WD 9.6 mm, mag100 K]. From the side [Figure 1(C), Working conditions: Acc.V 15.0 kV, WD 7.7 mm, mag70.0 K], tiny particles are distributed relatively regular on the walls of the nanotube, with no aggregation. Modification with or without PTES does not change the shape of Ag/TNT on SEM. XPS of Ag/TiO2 nanotube surfaces The chemical states of the elements in the Ag/TiO2 samples were analyzed by X-ray photoelectron spectroscopy (VGMultiLab 2000, Thermal Electron, USA) (Figure 2).After argon sputtering of 5 and 200 nm on Ag/TNT nanotubes, the atomic Ag content is 1.23 and 1.47%, respectively, which indicates Ag in the deep part of the tube. The highresolution XPS spectrum confined to the Ag window [Figure 2(C)] gave binding energies of Ag3d doublet peaks located at 368.5 (Ag3d5/2) and 374.5 (Ag3d3/2) eV. This reveals that the Ag exists in a metallic form.20 Surface wettability of super-hydrophobic Ag/TiO2 nanotube samples Contact angle measurements were performed at room temperature using a contact angle system (OCA 20, Data Physics, Germany). When water droplets are dropped onto the surface of TNT or Ag/TNT nanotubes [Figure 3(A,B)], the spherical droplets become flattened rapidly and scattered, wetting the surface. The contact angle of the measured surface is < 908, so the surface is hydrophilic. However, when water droplets are dropped onto the surfaces of S-Ag/ TNT nanotubes [Figure 3(C)], the droplets does not cling to the surface but slip down quickly or bounce up. When water droplets staying on the surface, their shapes remained basically the same; all measured contact angles are >1508, so the surfaces are super-hydrophobic. Effect of super-hydrophobic surface on Ag release The amount of Ag released from the Ag/TNT or S-Ag/TNT samples is measured by ICP-MS (ELAN 6100, Perkin-Elmer, Waltham, MA). There were three samples included in each group, the average amount of Ag released from Ag/TNT nanotubes on day 1 after treatment peaked at 0.3418 6 0.029 ppm
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FIGURE 1. SEM and energy-dispersive X-ray spectrometry (EDS) of the surface of Ag/TiO2 nanotubes. SEM of TiO2 nanotubes in frontal view (A); SEM of Ag/TiO2 nanotubes in frontal view (B) and profile view (C). D: EDS analysis of Ag/TiO2 nanotubes.
FIGURE 2. X-ray photoelectron spectroscopy analysis of Ag/TiO2 nanotubes; A: 5-mm sputtering depth; B: 200-mm sputtering depth. C: The high-resolution spectrum for the Ag 3d states.
but decreased to 0.2569 6 0.0312 ppm on day 1 after the sample super-hydrophobic modified (Figure 4). The percentage release on day 1 after modification among the total release volume in 14 days decreases from 30.7 to 23.1%, so super-hydrophobic modification inhibits the initial burst release of Ag on the surface. In addition, the Ag released on
days 2 and 3 is higher for S-Ag/TNT than Ag/TNT nanotubes, and still the amount of Ag release remain approximately 0.1 ppm on day 3, so super-hydrophobic modification has a certain controlled-release effect on Ag release. However, after day 5, each sample maintains relatively low release of Ag, so the Ag release rules of the 2 groups are basically the same.
FIGURE 3. Surface wettability of Ag/TiO2 nanotubes; A: TNT group; B: Ag/TNT group; C: S-Ag/TNT group.
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FIGURE 4. Silver release of Ag from Ag/TNT group and S-Ag/TNT nanotubes group.
In vitro anti-bacteria test for S-Ag/TNT nanotube surface After 2-h incubation, the amount of bacteria is the greatest on the TNT nanotube surface, followed by the Ag/TNT surface (Figure 5); the bacteria amount is low on the surfaces of S-TNT and S-Ag/TNT nanotubes and surfaces of Ag/TNT and S-Ag/TNT nanotubes show an increased number of red phosphor dots, indicating dead S. aureus. The amount of bacteria adhering to the surface of TNT nanotubes increases with time, and green phosphor dots are connected to form blocks or flakes. However, the bacteria are isolated and scattered on the super-hydrophobic surface; especially the S-Ag/TNT surface shows few bacteria and most of them are dead. The number of live bacteria with green fluorescent was different in each group [p < 0.05; Figure 5(C)]. On SEM, S. aureus bacteria cluster to form a typical botryoid shape on the TNT surface after 4-hr incubation [Figure 5(D,E)]; on the Ag/TNT surface, bacteria are dispersed, and the shapes are incomplete [Figure 5(F)]. The S-Ag/TNT surface shows only a small amount of
FIGURE 5. Bacterial adhesion in Ag/TiO2 nanotubes. A: Bacterial fluorescence staining of TNT group and S-TNT group at 2, 4 h, respectively. B: Bacterial fluorescence staining of Ag/TNT group and S-Ag/TNT group at 2, 4 h, respectively. C: Living bacterial counting under a fluorescence microscopy, the number of live bacteria with green fluorescent was different in each group (p < 0.05). D: Low magnification of SEM image, bacterial morphology of TNT group; E: High magnification of SEM image, bacterial morphology of TNT group; F: Low magnification of SEM image, bacterial morphology of Ag/TNT group. G: D: Low magnification of SEM image, bacterial morphology of S-Ag/TNT group.
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FIGURE 6. Antibacterial features of Ag/TiO2 nanotubes in New Zealand white rabbits. A: Radionuclide bone scan of 2 weeks postoperative (ROI 1 indicate TNT group, ROI 2 indicate S-Ag/TNT group, ROI 3 indicate normal bone tissue). B: The three groups show differences in radioactivity counting (p < 0.05). C: X-ray examination 2 weeks Postoperativly. D: X-ray examination 4 weeks postoperatively.
bacteria, with incomplete shapes; they are considered to be dead adhering bacteria after observation by fluorescence microscopy. In vivo anti-bacteria test of Ag/TNT and S-Ag/TNT titanium rods in rabbits Radionuclide bone scanning and radiography of Ti nanotubes in rabbits. During the test, all rabbits are allowed to move freely, and no animal dies. Rabbits implanted with TNT, Ag/TNT, and S-Ag/TNT nanotubes all show nonspecific inflammation at postoperative week 1 and mild radioactive concentration to different extents [Figure 6(A)]. At postoperative week 2, the TNT group shows radioactive concentration [Figure 6(A), ROI 1]] and local soft tissue swelling, which are considered an early manifestation of osteomyelitis. In both Ag/ TNT and S-Ag/TNT groups, inflammation are mild at postoperative week 2, indicating mild radioactive concentration [Figure 6(A), ROI 2]. The three groups show differences in radioactivity counting [p < 0.05; Figure 6(B)]. At postoperative week 2, radiography reveals local soft tissue swelling in the TNT group, but no fuzzy trabecular bone or manifestations of acute osteomyelitis such as bone destruction [Figure 6(C)]; neither Ag/TNT nor S-Ag/TNT groups shows soft tissue swelling. At postoperative week 4, distal femurs present osteoporosis and fuzzy trabecular bone with TNT treatment and osteomyelitis manifestations such as mild periosteal reaction; radiographs are normal with both Ag/TNT and S-Ag/TNT treatments.
Bacterial culture of eluent from Ti nanotubes in rabbits. TNT nanotube implantation produces a great number of bacteria, whereas Ag/TNT nanotubes produce fewer bacterial cultures (Figure 7). With S-Ag/TNT treatment, positive bacterial cultures are few and almost absent. Histology of Ti nanotubes from rabbits. Bone trabeculae are loose around TNT nanotubes at postoperative week 4, with bone destruction [Figure 8(A)]; no complete fibrous calluses are formed. A large number of inflammatory cells are visible in the medullary cavity, and a large amount of polynuclear phagocytes are visible on high magnification [Figure 8(E)]. Neither Ag/TNT nor S-Ag/TNT implants shows inflammation, and a large number of fibrous bone calluses or new bones are formed around the implant [Figure 8(B–D)]. Also, the bone interface integration is good, and cartilage cells proliferation are active around the bone interface on high magnification [Figure 8(F)]; the surrounding material generates abundant collagen. DISCUSSIONS
In general, super-hydrophobic coating can be prepared by two methods: constructing or modifying an appropriate dual micro/nanostructure on the material with low surface energy. The second method is always adopted for preparing a superhydrophobic surface for metallic orthopaedic implants. The
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FIGURE 7. Bacterial culture of eluent of Ag/TiO2 nanotubes from New Zealand white rabbits. A: TNT group; B: Ag/TNT group; C: S-Ag/TNT group.
FIGURE 8. Histology of Ag/TiO2 nanotubes from New Zealand white rabbits at 4 weeks after implantation. A: TNT group (3200); B: Ag/TNT group (3200); C: S-Ag/TNT group (3200). D: Fibrous bone calluses or new bone formed around S-Ag/TNT implant (340); E: Inflammation around TNT implant and a large amount of polynuclear phagocytes (arrow, 3400). F: New formed fibrous bone calluses around S-Ag/TNT implant (arrow, 3400).
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general techniques include the following methods: anodic oxidation,21,22 electrochemical deposition,23 plasma and laser etching,24 templating,25 and sol–gel.26 In this study, we use anodic oxidation and electrochemical deposition to create Ag/ TiO2 nanotubes. The prepared super-hydrophobic coatings on Ag/TiO2 nanotubes are regularly arranged; and nano-Ag particles (10–30 nm) are evenly distributed on the surface or inside nanotubes. The contact angles of the superhydrophobic-modified Ag/TiO2 nanotubes are all >1508. The super-hydrophobic character has a certain inhibitory effect on the burst release of Ag, for stable Ag release. Furthermore, the super-hydrophobic-coated Ag/TiO2 nanotubes are effective in inhibiting bacterial adhesion, killing the adhering bacteria, and preventing postoperative infection in vivo in rabbits. We prepare the TiO2 nanotube array membrane solidly connected to the base and regularly arranged with diameter 80–100 nm and length 200–400 nm as a carrier layer on the surface of Ti metal by electrochemical oxidation. Pulse electrodeposition is then used to deposit the Ag nanoparticles regularly on the surface and inside the wall of the nanotube to form a level structure with double roughness composed of nanoparticles and nanotubes. The contact angle reaches 161.68 6 0.58 after modification with PTES. Because of the special structure of the PTES molecule C8F13H4Si (ORe)3, the hydrophilic moiety Si-(ORe)3 is anchored inside of the nanotube by the non-covalent bond forces such as hydrogen bonds and Van der Waals force during self-assembly, and the hydrophobic moiety C8F13 remains outside the tube to construct the super-hydrophobic surface with the nanotube array. Although the prepared superhydrophobic surface can withstand disinfection methods such as cobalt irradiation and shows good ability to inhibit bacterial adhesion during bacterial culture in vitro, if the micro-structure of the super-hydrophobic surface is jeopardized or its super-hydrophobicity is compromised, the bacteria can show relatively strong proliferation once they adhere to the surface. Therefore, a compound coating must be integrated with the super-hydrophobic ability to inhibit bacterial adhesion and with antibacterial properties. Scholars in the textile field are the first to introduce Ag, with excellent antibacterial properties, into a superhydrophobic surface to construct material with the double effects of super-hydrophobic inhibition of bacteria and antibacterial properties. Yazdanshenas et al.15 adopt chemical approaches to spread Ag nanoparticles regularly on the surface of textiles to form surfaces with double roughness. The contact angle of the surface reaches 1518 after modification with PTES; in vitro experiments reveal that superhydrophobic-modified Ag/textile can effectively inhibit bacteria: no bacteria live on the surface of the material and inhibition zones against both E. coli and S. aureus are formed. In this study, we prepare a super-hydrophobic Ag coating on the surface of Ti implants by electrochemical oxidation and pulse electro-deposition. In vitro, bacterial adhesion is observed 2 and 4 h after bacterial culture, which basically agrees with the clinical period of bacterial adhesion with implants (2–6 h after surgery, the immunity of the organism is the lowest and antibiotics have not yet exerted
their efficacy). Fluorescence staining reveals that as compared with the common surface (TNT, Ag/TNT), the amount of bacteria adhering to the super-hydrophobic surface (STNT, S-Ag/TNT) is smaller, which indicates the excellent performance of the super-hydrophobic surface to inhibit bacterial adhesion. In addition, Ag/TNT and S-Ag/TNT shows relatively strong antibacterial ability; with time, fewer live bacteria appeared on the surface, with increasing number of dead bacteria. Moreover, in vivo experiments with rabbits show that Ag/TNT and S-Ag/TNT can prevent osteomyelitis and postoperative infection to reduce osteomyelitis signs such as bone destruction. Another purpose for constructing a super-hydrophobic Ag surface is to control Ag release by the superhydrophobic surface. Slow release of medicine can be realized with the air captured by a super-hydrophobic surface. Grinstaff et al.16 prepare a drug-carrying super-hydrophobic grid by the electrostatic spinning method, with the surface contact angle reaching 1538. The in vitro release test of the anticancer–drug-carrying super-hydrophobic grid reveals that high surface-contact angles inhibit water permeation, thus preventing the drug from dissolving with water, to slow the drug release. In vitro testing shows that this drugcarrying super-hydrophobic grid is effective in resisting cancer cells for as long as 60 days. Wesley L. Storm et al.27 fabricate a NO-releasing superhydrophobic membranes that can extend NO release durations from 59 to 105 h. As we find that, our super-hydrophobic modification has a certain release-controlled effect on Ag in the initial period, especially inhibiting the initial burst release of Ag on the first day, for more stable Ag release. After day 5, each Ag/surface basically maintains a relatively low Ag release, with superhydrophobic modification not affecting the release. With our in vitro release test, the super-hydrophobicity of samples decrease: the shapes of water droplets on the superhydrophobic surface turn from approximately spherical into hemispherical, which indicates the decrease of the contact angle and the super-hydrophobic properties. However, few studies have investigated the use of super-hydrophobic material to control the slow-release of a drug, especially in the field of orthopedics. Further research is needed to examine how to effectively increase the bonding strength and stability of the super-hydrophobic surface with time and the use of simple, effective methods to construct superhydrophobic membrane layers with self-repairing capability. CONCLUSIONS
We investigate the effect of super-hydrophobic-coated Ag/ TiO2 nanotubes on Ag release and the inhibitory effect on bacterial adhesion and antibacterial properties. The superhydrophobic modification has a certain controlled-release effect on the initial burst release of Ag, for stable Ag release and the double effects of inhibiting bacterial adhesion and surface antibacterial ability. REFERENCES 1. Barthlott W, Neinhuis C. Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta 1997;202:1–8.
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silver-containing PHBV nanofibrous scaffolds for tissue engineering. Biomacromolecules 2010;11:1248–1253. Shateri Khalil-Abad M, Yazdanshenas ME. Superhydrophobic antibacterial cotton textiles. J Colloid Interface Sci 2010;351:293–298. Yohe ST, Colson YL, Grinstaff MW. Superhydrophobic materials for tunable drug release: Using displacement of air to control delivery rates. J Am Chem Soc 2012;134:2016–2019. Yohe ST, Herrera VL, Colson YL, Grinstaff MW. 3D superhydrophobic electrospun meshes as reinforcement materials for sustained local drug delivery against colorectal cancer cells. J Control Release 2012;162:92–101. Gong D, Grimes CA, Varghese OK, Hu WC, Singh RS, Chen Z, Dickey EC. Titanium oxide nanotube arrays prepared by anodic oxidation. J Mater Res 2001;16:3331–3334. Zhuang HF, Lin CJ, Lai YK, Sun L, Li J. Some critical structure factors of titanium oxide manotube array in its photocatalytic activity. Environ Sci Technol 2007;41:4735–4740. Lai YK, Zhuang HF, Xie KP, Gong DG, Tang YX, Sun L, Lin CJ, Chen Z. Fabrication of uniform Ag/TiO2 nanotube array structures with enhanced photoelectrochemical performance. N J Chem 2010;34:1335–1340. Wang H, Dai D, Wu XD. Fabrication of superhydrophobic surfaces on aluminum. Appl Surf Sci 2008;254:5599–5601. Wu W, Wang X, Wang D, Chen M, Zhou F, Liu W, Xue Q. Alumina nanowire forests via unconventional anodization and superrepellency plus low adhesion to diverse liquids. Chem Commun (Camb) 2009;9:1043–1045. Li M, Zhai J, Liu H, Song YL, Jiang L, Zhu DB. Electrochemical deposition of conductive superhydrophobic zinc oxide thin films. J Phys Chem B 2003;107:9954–9957. Fresnais J, Chapel JP, Poncin-Epaillard F. Synthesis of transparent superhydrophobic polyethylene surfaces. Surf Coat Technol 2006;200:5296–5305. Thieme M, Frenzel R, Schmidt S, Simon F, Hennig A, Worch H, Lunkwitz K, Scharnweber D. Generation of ultrahydrophobic properties of aluminium—A first step to self-cleaning transparently coated metal surfaces. Adv Eng Mater 2001;3:691–695. Attawia MA, Uhrich KE, Botchwey E, Fan M, Langer R, Laurencin CT. Cytotoxicity testing of poly(anhydride-co-imides) for orthopedic applications. J Biomed Mater Res 1995;29:1233–1240. Storm WL, Youn J, Reighard KP, Worley BV, Lodaya HM, Shin JH, Schoenfisch MH. Superhydrophobic nitric oxide-releasing xerogels. Acta Biomater 2014;10:3442–3448.
INHIBITORY EFFECT OF SUPER-HYDROPHOBICITY
Department of Orthopaedics, General Hospital of Chinese PLA, Beijing 100853, China State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China 3 Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China 2
Received 7 November 2014; revised 28 April 2015; accepted 2 May 2015 Published online 20 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33454 Abstract: The antibacterial properties of super-hydrophobic silver (Ag) on implant surface have not yet to be fully illuminated. In our study, we investigate the protective effects of super-hydrophobic coating of silver/titanium dioxide (Ag/TiO2) nanotubes against bacterial pathogens, as well as its pattern of Ag release. Ag/TiO2 nanotubes are prepared by a combination of electrochemical anodization and pulse electrodeposition. The super-hydrophobic coating is prepared by modifying the surface of Ag/TiO2 nanotubes with 1H, 1H, 2H, 2Hperfluorooctyl-triethoxysilane (PTES). Surface features and Ag release are examined by SEM, X-ray photoelectron spectroscopy, contact-angle measurement, and inductively coupled plasma-mass spectrometry (ICP-MS). The antibacterial activity of super-hydrophobic coating Ag/TiO2 nanotubes is investigated both in vitro and in vivo. Consequently, the superhydrophobic coating on Ag/TiO2 nanotubes shows a regularly
arranged structure; and nano-Ag particles (10–30 nm) are evenly distributed on the surface or inside the nanotubes. The contact angles of water on the super-hydrophobic coating Ag/ TiO2 nanotubes are all above 1508. In addition, the superhydrophobic character displays a certain conserved effect that contributes to the sustained release of Ag. The superhydrophobic Ag/TiO2 nanotubes are also effective in inhibiting bacterial adhesion, killing the adhering bacteria and preventing postoperative infection in rabbits. Therefore, it is expected that the super-hydrophobic Ag/TiO2 nanotubes which can contain the release of Ag, leading to stable release, may show a conC 2015 Wiley Periodicals, sistent surface antibacterial capability. V Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1004–1012, 2016.
Key Words: Ag/TiO2 nanotubes, super-hydrophobic, silver release, bacterial adhesion, antibacterial activity
How to cite this article: Zhang L, Zhang L, Yang Y, Zhang W, Lv H, Yang F, Lin C, Tang P. 2016. Inhibitory effect of superhydrophobicity on silver release and antibacterial properties of super-hydrophobic Ag/TiO2 nanotubes. J Biomed Mater Res Part B 2016:104B:1004–1012.
INTRODUCTION
Since the 1990s, when Barthlott et al.1 clarify the selfcleaning effect of the surface of lotus leaves and the superhydrophobic phenomenon, super-hydrophobic surfaces have attracted wide attention.2–4 The super-hydrophobicity of lotus leaves results from the micro-nano structure of their surfaces: the surface of every micrometer mastoid is attached to numerous nanoscale wax crystal particles. This micro-nano structure can significantly increase the contact angle of water droplets on the surface so that they easily fall off. Hydrophilcity or hydrophobicity of a surface affects protein and bacterial adhesion.5 A hydrophilic surface (contact angle < 908) can accelerate the adhesion of bacteria such as
Staphylococcus aureus and Escherichia coli; however, a hydrophobic surface (contact angle 90–1508) can inhibit the adhesion of Deinococcus geothermalis and Meiothermus silvanus6; as well, S. aureus and Streptococcus mutans cannot easily adhere to the surface of hydrophobic materials.7,8 Thus, bacterial adhesion can be inhibited and infection risk reduced with super-hydrophobic coated materials. Only a few reports have shown the inhibitory effect of super-hydrophobic coating on bacterial adhesion.9–12 Poncin-Epaillard et al.11 fabricate a highly hydrophobic polymeric surface that can reduce the adhesion of both Gram positive and negative pathogenic bacteria. Schoenfisch et al.9 synthesize a super-hydrophobic gel coating that can
Additional Supporting Information may be found in the online version of this article. *These authors contributed equally to this work. Correspondence to: P. Tang; e-mail: [email protected] or C. Lin; e-mail: [email protected] Contract grant sponsor: The National Natural Science Foundation of China; contract grant number: 81171690 Contract grant sponsor: PLA Postgraduate Medical School; contract grant number: 11BCZ02
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significantly inhibit adhesion by S. aureus and Pseudomonas aeruginosa. Crick et al.10 prepare an elastic superhydrophobic coating that can inhibit the adhesion of E. coli and methicillin-resistant S. aureus. The super-hydrophobic surface can inhibit bacterial adhesion but does not kill bacteria; whether an antibacterial substance can be incorporated into a super-hydrophobic surface is unknown. Silver (Ag) has been widely used in every field of biomedicine because of its broad antibacterial spectrum, excellent thermal stability and desirable biological safety.13 Ag has powerful antibacterial properties and the lowest toxicity among the metal antibacterial agents.14 Therefore, introducing Ag with its enhanced antibacterial properties into a super-hydrophobic surface can be used to produce a superior antibacterial super-hydrophobic surface. Superhydrophobic Ag cotton fabrics successfully developed by Yazdanshenas et al.15 can effectively inhibit bacterial adhesion, and the Ag ion on the surface can kill a few E. coli and S. aureus bacteria adhering to and around the surface, for excellent antibacterial performance. However, no study has investigated the antibacterial effects of a super-hydrophobic Ag surface in biomedicine. As well, air captured by the super-hydrophobic surface can reduce the contact area between the material and body fluid as well as slow the speed of drug release,16,17 which can provide long-term release of Ag. In this study, we create Ag-supported/TiO2 nanotubes by a combination of electrochemical anodization and pulse electrodeposition and prepare a super-hydrophobic coating by modifying the surface of the Ag/TiO2 nanotubes with 1H, 1H, 2H, 2H-perfluorooctyl-triethoxysilane (PTES). We evaluate the inhibitory effect of the coating on bacterial adhesion and killing adherent bacteria and assess its effect on Ag release. METHODS
Preparation of coated Ag/TiO2 nanotubes Titanium (Ti) foil (99.6% pure Ti) is cut into 10 3 10 3 0.1 mm3. Titanium rods are cut into cylindrical geometry of 30 mm in length and 2.5 mm in diameter. Samples are polished by sandpaper, cleaned by ultrasonic vibration, then TiO2 nanotubes were fabricated in 0.5 wt % HF (Hydrofluoric acid) electrolyte using the previously reported electrochemical anodizing technique.18,19 Titanium sample was connected to the power supply as anode, and platinum plate electrode was selected as the cathode. Prepared samples are cleaned by ultrasonic vibration and placed after drying in a muffle furnace at constant temperature of 4508C for 2 h, to transform the amorphous TiO2 into anatase. Pulse electrodeposition is used to deposit Ag in the formed TiO2 nanotubes.20 At room temperature, nano-Ag particles deposition was carried out in an aqueous solution of 10 mM AgNO3 (silver nitrate) and 100 mM NaNO3 (sodium nitrate). The TiO2 nanotube sample and Pt plate were used as the working and counter electrodes, respectively. Nano-Ag particles are synthesized in a electrochemical workstation (Autolab PGSTAT 30, Eco Echemine BV Company, The Netherlands) with current density 10 mA cm22 and an alternating
procedure of opening every 0.1 s and closing every 0.3 s as well as deposition time 300 circles (cycle) to produce Ag/ TiO2 nanotubes (Ag/TNT). Samples are cleaned by ultrasonic vibration and preserved in a vacuum after drying. Test of physicochemical characteristics of coated Ag/ TiO2 nanotubes Field Emission Scanning Electron Microscope (Hitachi S4800, Hitachi High-Technologies Corporation, Japan) was employed to characterize the surface morphology of the Ag/ TiO2 Nanotubes. The Ag concentration of the surface of Ag/ TiO2 Nanotubes was determined by energy-dispersive X-ray spectroscopy (Hitachi S-4800, Hitachi High-Technologies Corporation, Japan). X-ray photoelectron spectrometer was utilized to determine the Ag distribution in the Ag/TiO2 nanotubes (VGMultiLab 2000, Thermal Electron, USA), with Al Ka as the radiation source and C1s 5 284.8 eV as the base of binding energy. Preparation of the super-hydrophobic coating of Ag/ TiO2 nanotubes Samples (TNT, Ag/TNT) are soaked in methanol solution with 1% 1H, 1H, 2H, 2H-perfluorooctyl-triethoxysilane (PTES; Degussa) for 1 h, and then placed in a drying oven at 1408C for 1 h. The PTES-modified sample is recorded as super-hydrophobic TiO2 nanotubes (S-TNT) and superhydrophobic Ag/TiO2 nanotubes (S-Ag/TNT). Samples are disinfected by irradiation with cobalt-60, and then the surface wettability was evaluated by measuring the contact angle between the sample and water droplets. Contact angle measurements were performed at room temperature using a Contact angle system (OCA 20, Data Physics, Germany). Effect of super-hydrophobic property on Ag release The amount of Ag released from the Ag/TNT or S-Ag/TNT samples was monitored in phosphate buffered saline (0.01 M PBS) at 378C. The samples were immersed in 6 mL of PBS for 24 h in the thermostated container, taken out, and then immersed again in 6 mL of fresh PBS. This process is repeated for 14 days. The concentration of Ag released is measured by ICP-MS (ELAN 6100, Perkin-Elmer, Waltham, MA). In vitro antimicrobial test of Ag/TNT and S-Ag/TNT Samples (TNT, S-TNT, Ag/TNT, S-Ag/TNT) are placed in sterile 24-well plates, and 2 mL S. aureus 8325 solution (1 3 106 colony forming units [cfu]/mL) is placed onto the surface of each sample. Plates are placed in an incubator with relative humidity > 90% and temperature 378C. After 2 or 4 h, plates are removed and washed with sterile PBS to remove non-adhering bacteria. Some samples are stained with LIVE/DEAD fluorescence staining reagent (Molecular Probes). After samples are stained away from the light for 15 min, they are washed with PBS. The distribution of bacteria was observed under a fluorescent microscope, and bacteria with green fluorescent (live bacteria) were counted in a field 200 3 200 mm2 by use of Image-Pro (MediaCybernetics, USA). Six views of TNT, S-TNT, Ag/TNT, S-Ag/TNT were selected for counting. Samples are fixed with 2.5%
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glutaral solution, kept in a 48C refrigerator overnight, dehydrated and critical point drying, and then spraying the surface of the sample thin gold film. At last, the samples are observed by SEM. In vivo antimicrobial test of S-Ag/TNT S-Ag/TNT implantation. We use 15 healthy male New Zealand white rabbits aged 8 months (body weight 2.5–3 kg) from the Laboratory Animal Center of PLA General Hospital. Total 30 hind legs are randomly divided into three groups (n 5 10 each): Titanium rods of TNT structured surfaces (TNT Group), Titanium rods of Ag/TNT structured surfaces (Ag/TNT Group) and Titanium rods of S-Ag/TNT structured surfaces (S-Ag/TNT Group). The animals are anesthetized with a 0.3 mL kg21 mixture of ketamine and sumianxin via intramuscular injection, areas are disinfected, and a sterile towel is applied. A vertical incision parallel to the femur is made at the lower end of femur and lateral knee joint, and skin, subcutaneous tissue and medial patellar ligament are cut away layer by layer, before the articular cavity is opened to disclose the femoral condyle. At 0.5 cm over the cross point of the femoral axis and the connection line of bilateral femoral condyles, bone tissue is reamed toward the proximal femur successively with use of Kirschner wire and different types of drill bits, before the medullary cavity is flushed with normal saline. An amount of 1 3 104 cfu mL21 S. aureus liquid (20 lL) is injected into the medullary cavity, before the TNT samples are implanted into the medullary cavity of distal femur, and the opening at the femoral condyle is closed with use of bone wax. The articular cavity is flushed with normal saline, and closed, before the incision is sutured layer by layer. After surgery, no antibiotic treatment is applied, and the incision is wiped with ethanol every day during postoperative 1 week. Postoperative radiography. Radionuclide bone scanning of bilateral femurs is conducted at postoperative weeks 1 and 2 for 2 to 4 h after injection of 99Tc-MDP, 10 mG, with acquisition interval (upper collection distance) 3 cm. The region of interest is at the symmetrical area of bilateral lower femurs. Radiographs are taken at postoperative weeks 2 and 4. Bacterial culture of bacteria eluent from implants. After animals are anesthetized by an overdose of ketamine and sumianxin and sacrificed, bilateral femurs are excised under aseptic conditions. At 3 cm from the screw entrance point at the femoral condyle, femurs are sawed off transversely to take out the nanotube. The surface of the nanotube is washed with normal saline solution to remove excess blood or bacteria, before cleaned by ultrasonography in 2 mL normal saline to remove adhered bacteria. Bacterial eluent removed is used for culture. Histology. Distal femoral bone specimens are fixed with 10% neutral formalin for 3 days, decalcification with 10% EDTA, embedded in paraffin and sectioned, before undergo hematoxylin and eosin (H&E) and Masson trichrome staining.
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Statistical analysis Data are described with mean 6 SD. Differences between groups are compared by one-way ANOVA and Student–Newman–Keuls test. Analysis involves use of StatView (SAS, Cary, NC). p < 0.05 is considered statistically significant. RESULTS
SEM and EDS of Ag/TiO2 nanotubes Pulse-electrodeposited samples are analyzed by SEM and EDS [Figure 1(D)]. The EDS spectral peaks (Working conditions: Acc.V 15.0 KV, mag400) reveal 3 elements: Ti, O, and Ag. After Ag is introduced onto the surface of samples by pulse electrodeposition, the shape of the tube does not change as compared with the pure TiO2 nanotube [Figure 1(A) and supplymentary Figure 1, Working conditions: Acc.V 20.0 KV, WD 10.5 mm, mag100 K]; however, oval or round particles of 10–30 nm are regularly distributed on the surface and at the orifice of TiO2 nanotubes [Figure 1(B), Working conditions: Acc.V 15.0 kV, WD 9.6 mm, mag100 K]. From the side [Figure 1(C), Working conditions: Acc.V 15.0 kV, WD 7.7 mm, mag70.0 K], tiny particles are distributed relatively regular on the walls of the nanotube, with no aggregation. Modification with or without PTES does not change the shape of Ag/TNT on SEM. XPS of Ag/TiO2 nanotube surfaces The chemical states of the elements in the Ag/TiO2 samples were analyzed by X-ray photoelectron spectroscopy (VGMultiLab 2000, Thermal Electron, USA) (Figure 2).After argon sputtering of 5 and 200 nm on Ag/TNT nanotubes, the atomic Ag content is 1.23 and 1.47%, respectively, which indicates Ag in the deep part of the tube. The highresolution XPS spectrum confined to the Ag window [Figure 2(C)] gave binding energies of Ag3d doublet peaks located at 368.5 (Ag3d5/2) and 374.5 (Ag3d3/2) eV. This reveals that the Ag exists in a metallic form.20 Surface wettability of super-hydrophobic Ag/TiO2 nanotube samples Contact angle measurements were performed at room temperature using a contact angle system (OCA 20, Data Physics, Germany). When water droplets are dropped onto the surface of TNT or Ag/TNT nanotubes [Figure 3(A,B)], the spherical droplets become flattened rapidly and scattered, wetting the surface. The contact angle of the measured surface is < 908, so the surface is hydrophilic. However, when water droplets are dropped onto the surfaces of S-Ag/ TNT nanotubes [Figure 3(C)], the droplets does not cling to the surface but slip down quickly or bounce up. When water droplets staying on the surface, their shapes remained basically the same; all measured contact angles are >1508, so the surfaces are super-hydrophobic. Effect of super-hydrophobic surface on Ag release The amount of Ag released from the Ag/TNT or S-Ag/TNT samples is measured by ICP-MS (ELAN 6100, Perkin-Elmer, Waltham, MA). There were three samples included in each group, the average amount of Ag released from Ag/TNT nanotubes on day 1 after treatment peaked at 0.3418 6 0.029 ppm
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FIGURE 1. SEM and energy-dispersive X-ray spectrometry (EDS) of the surface of Ag/TiO2 nanotubes. SEM of TiO2 nanotubes in frontal view (A); SEM of Ag/TiO2 nanotubes in frontal view (B) and profile view (C). D: EDS analysis of Ag/TiO2 nanotubes.
FIGURE 2. X-ray photoelectron spectroscopy analysis of Ag/TiO2 nanotubes; A: 5-mm sputtering depth; B: 200-mm sputtering depth. C: The high-resolution spectrum for the Ag 3d states.
but decreased to 0.2569 6 0.0312 ppm on day 1 after the sample super-hydrophobic modified (Figure 4). The percentage release on day 1 after modification among the total release volume in 14 days decreases from 30.7 to 23.1%, so super-hydrophobic modification inhibits the initial burst release of Ag on the surface. In addition, the Ag released on
days 2 and 3 is higher for S-Ag/TNT than Ag/TNT nanotubes, and still the amount of Ag release remain approximately 0.1 ppm on day 3, so super-hydrophobic modification has a certain controlled-release effect on Ag release. However, after day 5, each sample maintains relatively low release of Ag, so the Ag release rules of the 2 groups are basically the same.
FIGURE 3. Surface wettability of Ag/TiO2 nanotubes; A: TNT group; B: Ag/TNT group; C: S-Ag/TNT group.
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FIGURE 4. Silver release of Ag from Ag/TNT group and S-Ag/TNT nanotubes group.
In vitro anti-bacteria test for S-Ag/TNT nanotube surface After 2-h incubation, the amount of bacteria is the greatest on the TNT nanotube surface, followed by the Ag/TNT surface (Figure 5); the bacteria amount is low on the surfaces of S-TNT and S-Ag/TNT nanotubes and surfaces of Ag/TNT and S-Ag/TNT nanotubes show an increased number of red phosphor dots, indicating dead S. aureus. The amount of bacteria adhering to the surface of TNT nanotubes increases with time, and green phosphor dots are connected to form blocks or flakes. However, the bacteria are isolated and scattered on the super-hydrophobic surface; especially the S-Ag/TNT surface shows few bacteria and most of them are dead. The number of live bacteria with green fluorescent was different in each group [p < 0.05; Figure 5(C)]. On SEM, S. aureus bacteria cluster to form a typical botryoid shape on the TNT surface after 4-hr incubation [Figure 5(D,E)]; on the Ag/TNT surface, bacteria are dispersed, and the shapes are incomplete [Figure 5(F)]. The S-Ag/TNT surface shows only a small amount of
FIGURE 5. Bacterial adhesion in Ag/TiO2 nanotubes. A: Bacterial fluorescence staining of TNT group and S-TNT group at 2, 4 h, respectively. B: Bacterial fluorescence staining of Ag/TNT group and S-Ag/TNT group at 2, 4 h, respectively. C: Living bacterial counting under a fluorescence microscopy, the number of live bacteria with green fluorescent was different in each group (p < 0.05). D: Low magnification of SEM image, bacterial morphology of TNT group; E: High magnification of SEM image, bacterial morphology of TNT group; F: Low magnification of SEM image, bacterial morphology of Ag/TNT group. G: D: Low magnification of SEM image, bacterial morphology of S-Ag/TNT group.
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FIGURE 6. Antibacterial features of Ag/TiO2 nanotubes in New Zealand white rabbits. A: Radionuclide bone scan of 2 weeks postoperative (ROI 1 indicate TNT group, ROI 2 indicate S-Ag/TNT group, ROI 3 indicate normal bone tissue). B: The three groups show differences in radioactivity counting (p < 0.05). C: X-ray examination 2 weeks Postoperativly. D: X-ray examination 4 weeks postoperatively.
bacteria, with incomplete shapes; they are considered to be dead adhering bacteria after observation by fluorescence microscopy. In vivo anti-bacteria test of Ag/TNT and S-Ag/TNT titanium rods in rabbits Radionuclide bone scanning and radiography of Ti nanotubes in rabbits. During the test, all rabbits are allowed to move freely, and no animal dies. Rabbits implanted with TNT, Ag/TNT, and S-Ag/TNT nanotubes all show nonspecific inflammation at postoperative week 1 and mild radioactive concentration to different extents [Figure 6(A)]. At postoperative week 2, the TNT group shows radioactive concentration [Figure 6(A), ROI 1]] and local soft tissue swelling, which are considered an early manifestation of osteomyelitis. In both Ag/ TNT and S-Ag/TNT groups, inflammation are mild at postoperative week 2, indicating mild radioactive concentration [Figure 6(A), ROI 2]. The three groups show differences in radioactivity counting [p < 0.05; Figure 6(B)]. At postoperative week 2, radiography reveals local soft tissue swelling in the TNT group, but no fuzzy trabecular bone or manifestations of acute osteomyelitis such as bone destruction [Figure 6(C)]; neither Ag/TNT nor S-Ag/TNT groups shows soft tissue swelling. At postoperative week 4, distal femurs present osteoporosis and fuzzy trabecular bone with TNT treatment and osteomyelitis manifestations such as mild periosteal reaction; radiographs are normal with both Ag/TNT and S-Ag/TNT treatments.
Bacterial culture of eluent from Ti nanotubes in rabbits. TNT nanotube implantation produces a great number of bacteria, whereas Ag/TNT nanotubes produce fewer bacterial cultures (Figure 7). With S-Ag/TNT treatment, positive bacterial cultures are few and almost absent. Histology of Ti nanotubes from rabbits. Bone trabeculae are loose around TNT nanotubes at postoperative week 4, with bone destruction [Figure 8(A)]; no complete fibrous calluses are formed. A large number of inflammatory cells are visible in the medullary cavity, and a large amount of polynuclear phagocytes are visible on high magnification [Figure 8(E)]. Neither Ag/TNT nor S-Ag/TNT implants shows inflammation, and a large number of fibrous bone calluses or new bones are formed around the implant [Figure 8(B–D)]. Also, the bone interface integration is good, and cartilage cells proliferation are active around the bone interface on high magnification [Figure 8(F)]; the surrounding material generates abundant collagen. DISCUSSIONS
In general, super-hydrophobic coating can be prepared by two methods: constructing or modifying an appropriate dual micro/nanostructure on the material with low surface energy. The second method is always adopted for preparing a superhydrophobic surface for metallic orthopaedic implants. The
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FIGURE 7. Bacterial culture of eluent of Ag/TiO2 nanotubes from New Zealand white rabbits. A: TNT group; B: Ag/TNT group; C: S-Ag/TNT group.
FIGURE 8. Histology of Ag/TiO2 nanotubes from New Zealand white rabbits at 4 weeks after implantation. A: TNT group (3200); B: Ag/TNT group (3200); C: S-Ag/TNT group (3200). D: Fibrous bone calluses or new bone formed around S-Ag/TNT implant (340); E: Inflammation around TNT implant and a large amount of polynuclear phagocytes (arrow, 3400). F: New formed fibrous bone calluses around S-Ag/TNT implant (arrow, 3400).
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general techniques include the following methods: anodic oxidation,21,22 electrochemical deposition,23 plasma and laser etching,24 templating,25 and sol–gel.26 In this study, we use anodic oxidation and electrochemical deposition to create Ag/ TiO2 nanotubes. The prepared super-hydrophobic coatings on Ag/TiO2 nanotubes are regularly arranged; and nano-Ag particles (10–30 nm) are evenly distributed on the surface or inside nanotubes. The contact angles of the superhydrophobic-modified Ag/TiO2 nanotubes are all >1508. The super-hydrophobic character has a certain inhibitory effect on the burst release of Ag, for stable Ag release. Furthermore, the super-hydrophobic-coated Ag/TiO2 nanotubes are effective in inhibiting bacterial adhesion, killing the adhering bacteria, and preventing postoperative infection in vivo in rabbits. We prepare the TiO2 nanotube array membrane solidly connected to the base and regularly arranged with diameter 80–100 nm and length 200–400 nm as a carrier layer on the surface of Ti metal by electrochemical oxidation. Pulse electrodeposition is then used to deposit the Ag nanoparticles regularly on the surface and inside the wall of the nanotube to form a level structure with double roughness composed of nanoparticles and nanotubes. The contact angle reaches 161.68 6 0.58 after modification with PTES. Because of the special structure of the PTES molecule C8F13H4Si (ORe)3, the hydrophilic moiety Si-(ORe)3 is anchored inside of the nanotube by the non-covalent bond forces such as hydrogen bonds and Van der Waals force during self-assembly, and the hydrophobic moiety C8F13 remains outside the tube to construct the super-hydrophobic surface with the nanotube array. Although the prepared superhydrophobic surface can withstand disinfection methods such as cobalt irradiation and shows good ability to inhibit bacterial adhesion during bacterial culture in vitro, if the micro-structure of the super-hydrophobic surface is jeopardized or its super-hydrophobicity is compromised, the bacteria can show relatively strong proliferation once they adhere to the surface. Therefore, a compound coating must be integrated with the super-hydrophobic ability to inhibit bacterial adhesion and with antibacterial properties. Scholars in the textile field are the first to introduce Ag, with excellent antibacterial properties, into a superhydrophobic surface to construct material with the double effects of super-hydrophobic inhibition of bacteria and antibacterial properties. Yazdanshenas et al.15 adopt chemical approaches to spread Ag nanoparticles regularly on the surface of textiles to form surfaces with double roughness. The contact angle of the surface reaches 1518 after modification with PTES; in vitro experiments reveal that superhydrophobic-modified Ag/textile can effectively inhibit bacteria: no bacteria live on the surface of the material and inhibition zones against both E. coli and S. aureus are formed. In this study, we prepare a super-hydrophobic Ag coating on the surface of Ti implants by electrochemical oxidation and pulse electro-deposition. In vitro, bacterial adhesion is observed 2 and 4 h after bacterial culture, which basically agrees with the clinical period of bacterial adhesion with implants (2–6 h after surgery, the immunity of the organism is the lowest and antibiotics have not yet exerted
their efficacy). Fluorescence staining reveals that as compared with the common surface (TNT, Ag/TNT), the amount of bacteria adhering to the super-hydrophobic surface (STNT, S-Ag/TNT) is smaller, which indicates the excellent performance of the super-hydrophobic surface to inhibit bacterial adhesion. In addition, Ag/TNT and S-Ag/TNT shows relatively strong antibacterial ability; with time, fewer live bacteria appeared on the surface, with increasing number of dead bacteria. Moreover, in vivo experiments with rabbits show that Ag/TNT and S-Ag/TNT can prevent osteomyelitis and postoperative infection to reduce osteomyelitis signs such as bone destruction. Another purpose for constructing a super-hydrophobic Ag surface is to control Ag release by the superhydrophobic surface. Slow release of medicine can be realized with the air captured by a super-hydrophobic surface. Grinstaff et al.16 prepare a drug-carrying super-hydrophobic grid by the electrostatic spinning method, with the surface contact angle reaching 1538. The in vitro release test of the anticancer–drug-carrying super-hydrophobic grid reveals that high surface-contact angles inhibit water permeation, thus preventing the drug from dissolving with water, to slow the drug release. In vitro testing shows that this drugcarrying super-hydrophobic grid is effective in resisting cancer cells for as long as 60 days. Wesley L. Storm et al.27 fabricate a NO-releasing superhydrophobic membranes that can extend NO release durations from 59 to 105 h. As we find that, our super-hydrophobic modification has a certain release-controlled effect on Ag in the initial period, especially inhibiting the initial burst release of Ag on the first day, for more stable Ag release. After day 5, each Ag/surface basically maintains a relatively low Ag release, with superhydrophobic modification not affecting the release. With our in vitro release test, the super-hydrophobicity of samples decrease: the shapes of water droplets on the superhydrophobic surface turn from approximately spherical into hemispherical, which indicates the decrease of the contact angle and the super-hydrophobic properties. However, few studies have investigated the use of super-hydrophobic material to control the slow-release of a drug, especially in the field of orthopedics. Further research is needed to examine how to effectively increase the bonding strength and stability of the super-hydrophobic surface with time and the use of simple, effective methods to construct superhydrophobic membrane layers with self-repairing capability. CONCLUSIONS
We investigate the effect of super-hydrophobic-coated Ag/ TiO2 nanotubes on Ag release and the inhibitory effect on bacterial adhesion and antibacterial properties. The superhydrophobic modification has a certain controlled-release effect on the initial burst release of Ag, for stable Ag release and the double effects of inhibiting bacterial adhesion and surface antibacterial ability. REFERENCES 1. Barthlott W, Neinhuis C. Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta 1997;202:1–8.
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INHIBITORY EFFECT OF SUPER-HYDROPHOBICITY
