Long-lasting in vivo and in vitro antibacterial ability of nanostructured titania coating incorporated with silver nanoparticles Hao Cheng,1 Yong Li,1 Kaifu Huo,2 Biao Gao,2 Wei Xiong1 1
Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China 2 School of Materials and Metallurgy, Wuhan University of Science and Technology, Wuhan 430081, China Received 9 August 2013; revised 5 October 2013; accepted 23 October 2013 Published online 15 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35019 Abstract: Although titanium (Ti) implants are widely used clinically, implant-associated bacterial infection is still one of the most serious complications in orthopedic surgery. Long-term antibacterial properties and the ability to inhibit biofilm formation are highly desirable to prevent implant associated infection. In this study, a controllable amount of silver (Ag) nanoparticles was incorporated into titanium oxide; or titanium, nanotubes (TiO2-NTs). The reliable release and long-term antibacterial function of Ag, in vivo and in vitro, and influence normal boneimplant integration from the Ag released from Ag-incorporated NTs in vivo have been studied to make them useable in clinical practice. In the current study, TiO2-NTs loaded with Ag (NT-Ag) exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) in vitro for 30 days,
and the ability to penetrate the protein layer well. In addition, Xray examination and 2-[18F]-fiuoro-2-deoxy-D-glucose positron emission tomography indicates that NT-Ag show extremely long antibacterial activity in vivo in a rat model. Furthermore, histomorphometric analysis demonstrated that satisfactory biointegration can be expected. Our results indicate that NT-Ag has both simultaneous antimicrobial and excellent bio-integration properties, make it a promising therapeutic material for orthopeC 2013 Wiley Periodicals, Inc. J Biomed Mater Res dic application. V Part A: 102A: 3488–3499, 2014.
Key Words: implant-associated infections, rat model, long antibacterial activity, silver nanoparticle, titania nanotubes (TiO2-NTs)
How to cite this article: Cheng H, Li Y, Huo K, Gao B, Xiong W. 2014. Long-lasting in vivo and in vitro antibacterial ability of nanostructured titania coating incorporated with silver nanoparticles. J Biomed Mater Res Part A 2014:102A:3488–3499.
INTRODUCTION
Postoperative infection associated with implants remains one of the most devastating complications in orthopedic surgery, sometimes demanding implant removal and/or repeated surgeries resulting in huge substantial costs and extended hospitalization. Usually these infections are difficult to diagnose in the early stages, which allows the invading bacteria to form a biofilm. This biofilm acts as a protective screen against antibacterial agents and host defense. In addition, the bacteria in a biofilm form will have a lower metabolic rate, making eradication, and detachment of the infective pathogens difficult, especially for growthdependent antibiotics.1–4 Antibiotic concentration can be up to 1000-fold higher needed to inhibit the growth of bacteria in biofilms compared to the planktonic bacteria.4,5–7 In addition, internalization of Staphylococcus aureus by osteoblast protects the bacteria from the host immune system,8–11 thus forming a reservoir of bacteria for recurring infection.12,13 By residing a longer time in vivo, the biofilms gradually become more resistant to antibiotics.14 Antibacterial coatings fabricated on the surfaces of biomedical devices can decrease the risk of the infection with
such bacteria by preventing microbe adhesion and proliferation. For such clinical applications, coatings must have combination of excellent antibacterial efficacy and low little toxicity to eukaryotic cells. Recently, significant attention has been given to antibacterial coatings containing silver (Ag), which has long been known to serve as an antibacterial agent that kills and inhibits a wide spectrum of microorganisms, including antibiotic-resistant bacteria, while possessing several other advantages such as satisfactory, stability, little possibility to develop antibiotic resistant strains, and non-cytotoxic at appropriate doses.1,15–21 In particular, Ag ions seems to show the best antibacterial activity.22–25 Although materials containing Ag have been widely explored for use in preventing biofilm formation, controlling the dosage and obtaining consistent therapeutic outcomes have been challenging. Titania nanotubes (TiO2-NTs) fabricated on titanium (Ti) implants by electrochemical anodization solves both of these issues.26–33 TiO2-NTs also offer the possibility of long-term antibacterial treatment time by controlling the release of nanoparticles such as Ag. For example, Ag release from TiO2-NTs can be regulated by the tube dimensions.34 The diameter of the TiO2-NTs and the space between nanotubes can be varied
Correspondence to: W. Xiong; e-mail: [email protected]
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from hundreds to tens of nanometers by adjusting several anodization parameters, thus, offering the most direct, feasible method for achieving the controlled release of Ag. Moreover, there is evidence that a range of competing chemical approaches can make the release of biologically active Ag controllable by changing the oxidation reactions in water.35 One of the other merits of this approach is that TiO2-NTs have a lower elastic modulus;32 thus, better biomechanical compatibility can be expected, compared to other artificial biomaterials, due to the close match with the elastic modulus of natural bone. TiO2-NTs induce mineralization, extracellular matrix secretion of nanostructured hydroxyapatite growing in vivo,28 and simultaneously promote osteogenic differentiation and mesenchymal stem cells proliferation.27,29–31 TiO2-NTs could act as carrier, which can store and release Ag ions in a controllable manner, showing long-term antibacterial capability. Generally speaking, TiO2-NTs are ideal bioactive coatings that can serve as carriers for Ag ions and other antibacterial agents, and simultaneously induce direct bone-implant bonding; however, clinical application additional in vivo studies for further verification. In this study, we investigate whether a proper quantity Ag incorporated into TiO2-NTs could be reliably released, and if the Ag loaded TiO2-NTs (NT-Ag) possesses long-term antibacterial function, both in vivo and in vitro. In addition, we studied the osseointegration property of NT-Ag in vivo. MATERIALS AND METHODS
Specimen preparation In brief, Ti foil (99.7% pure, Aldrich, 10 3 20 3 1 mm3) and titanium rods (diameter: 0.8 mm; length: 12 mm) were prepared. The TiO2-NTs were fabricated by electrochemical anodization, using a direct-current (DC) power supply (IT6834, ITECH, China). Ethylene glycol containing 0.5 wt% ammonium fluoride (NH4F) and 5 vol% distilled deionized (DI) water was used as an electrolyte. After anodization at 60 V for 1 h, amorphous TiO2-NTs (TiO2-NTAs) formed on the Ti foils. The amorphous form was converted into anatase TiO2-NTs by annealing at 450 C in air for 3 h. The anatase TiO2-NTs were soaked in AgNO3 solution (>99.8% purity) (H2O:C2H5OH 5 1:1) with a concentration of 1.5 M for 30 min. The samples were rinsed carefully with DI water and dried in an air stream. Then the samples were irradiated with ultraviolet light for 30 min using a highpressure Hg lamp. Surface characterization The surface morphology, topography, and structure of the TiO2-NTs and NT-Ag specimens were characterized by fieldemission scanning electron microscopy (FE-SEM, FEI Nova 400 Nano), transmission electron microscopy (TEM, Philips CM20), and atomic force microscopy (AFM, Auto-Probe CP, Park Scientific Instruments). Ag release The content of Ag released from the NT-Ag samples was monitored in phosphate-buffered saline (PBS) as follows.
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Firstly, the samples were immersed in 6 mL of PBS for 1 day in the dark and then taken out, and washed with DI water. The samples were dipped again in 6 mL of fresh PBS, and the process was repeated for a total 14 days to obtain the solution with released Ag1 content. The PBS solution containing released silver was analyzed by inductivelycoupled plasma-atomic emission spectrometry (ICP-AES, IRIS Advantage ER/S). Antibacterial assay Foil and rod samples were prepared as described earlier. The antibacterial activity was tested against methicillinresistant Staphylococcus aureus (MRSA, ATCC43300) cultivated in a trypticase soy broth (TSB) medium at 37 C for 18 h. It was diluted to a concentration of 105 CFU mL21 in the antibacterial assay. Each rod specimen and foil specimen was incubated in 1 mL of the bacteria suspension in TSB at 37 C for 1 day. The culture medium was sampled to determine the viable counts of planktonic bacteria. Non-adherent bacteria were eliminated by three gentle rinses with PBS. Then the each specimen was placed in 1 mL of TSB followed by ultrasonic vibration (40 W) for 5 min to detach the adhered bacteria. The resulting suspension was sampled to count the viable bacteria adhered to the specimens. The specimens were repeatedly used for a total incubation time of 30 days after ultrasonic cleaning, and sterilization by moist heat disinfection. The live bacteria in the sampled suspensions were counted using the serial dilution and the spread-plate method at days 1, 4, 7, 10, 15, 20, and 30. These sampling time points were chosen based on previous studies.36,37 The antibacterial effect on planktonic bacteria and adhered bacteria was represented by a bactericidal ratio, based on the following formulas: (1) the bactericidal ratio of planktonic bacteria growing in the TSB (Rap) (%) 5 (B2A) / A 3 100%, and (2) the bactericidal ratio of adhered bacteria on the surface of the specimens (Raa) (%) 5 (D2C) / C 3 100%, where A is the mean number of viable bacteria in the TSB medium inoculated with NT-Ag or TiO2-NTs, B is the mean number of viable bacteria in the TSB medium inoculated with pure titanium foils, C is the mean number of viable bacteria on the NT-Ag or TiO2-NT specimens, and D is the mean number of viable bacteria on the pure Ti foils. Zone of inhibition (ZOI) tests were performed in which 10 mL S. aureus, adjusted to 1.5 3 108 CFUs mL21, was spread evenly over Mueller–Hinton plates. The foil and rod samples were placed on the prepared plates separately. The plates were incubated at 37 C under anaerobic conditions for 24 h and photographed to record the results. Antiadhesive assay The antiadhesive effect of the surface of the samples was qualitatively tested by scanning electron microscopy (SEM; S-4800; Hitachi Tokyo, Japan). The samples were immersed 1 mL bacteria suspension in TSB, which contained 1.5 3 108 cells cultured for 24 h at 37 C. Non-adherent bacteria were removed by three gentle rinses with PBS. The samples were subsequently fixed with glutaraldehyde solution (2.5%
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concentration) for 2 h at 4 C. Then the samples were dehydrated in an ethanol series (30, 50, 70, 90, and 100%). After dehydration, an amyl acetate solution was used to replace the ethanol for 1 h at room temperature, and then the samples were critical-point dried. The adherent bacteria on resultant samples were coated with Au in a sputter coater and imaged using SEM. Animal surgery All surgeries in the in vivo experiment were performed under protocols approved by the Ethics Committee for Animal Experiments of Huazhong University of Science and Technology, Wu Han, China (permit number 10-07-02). The study used a total of 60 adult male SD rats (average weight: 150 g), supplied by the Laboratory Animal Center of Tongji Medical College of Huazhong University of Science and Technology. Thirty rats were equally divided into three groups and implanted with the three kinds of rod samples only for histomorphometry analysis: group I (Ti-rod implant), group II (TiO2-NT rod implant), and group III (NTAg rod implant). The other 30 rats, distributed into three groups, were injected with 2 lL bacteria and inserted with implants simultaneously. The rats were kept under specific pathogen free (SPF) conditions in our Laboratory Animal Center. The rats were anesthetized with 50 mg pentobarbital per kg body weight by intraperitoneal injection before surgery. Details regarding the surgical procedure are described in a previous report.38 After shaving and sterilizing the site with povidone iodine, an incision was made across the left knee. A sterilized syringe needle (external diameter: 0.8 mm) was used to drill a hole through the tibial plateau, from the proximal end to the distal end. After the medullary cavity was inoculated with 1 lL bacteria suspension in TSB (which contained 1.5 3 108 cells mL21), the three different types of rod samples were implanted into the medullary cavity. The other 30 rats were implanted with the three types of rod implants only. The burr hole was sealed with bone wax; skin openings were closed by sutures and sterilized with povidone iodine. PET examination For 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET), the 30 rats injected with 1 lL bacteria were injected with 93 MBq 18F-FDP through the caudal vein on day 5 and day 10 after surgery. Then the rats were anesthetized as described earlier, and 60 min after injection of the 18F-FDG, PET (Discovery LS PET/CT system, GE) was performed. In vivo radiology X-ray examination (Kodak, Directview, DR3000) was performed 2, 3, and 4 weeks after surgery to assess longitudinal osteolysis of the rats. Histological analysis and immunohistochemistry Three rats in each group were euthanized anesthesia, with an overdose of pentobarbital at 2, 3, and 4 weeks. The left tibia was removed and separated from the soft tissue, and
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subsequently fixed in 4% paraformaldehyde for 12 h. Then the specimens were incubated in ethylenediaminetetraacetic acid (EDTA) and embedded in paraffin. Sagittal sections were cut into sections (5 lm thick) and then were stained with hematoxylin and eosin (H&E). The sagittal sections were used for immunostaining. An indirect immunostaining technique, based on a specific S. aureus murine monoclonal antibody (ab37644; abcam plc, Cambridge, UK), was used for the in situ identification of S. aureus. Antigen retrieval was performed using a microwave repair treatment for 15 min after deparaffinization. Blocking of endogenous peroxidase activity was performed by placing the slides in 5% H2O2 for 10–30 min. After the addition of 5% bovine serum albumin (BSA), the reaction was developed for 30 min at room temperature. Then the specific S. aureus murine monoclonal antibody was added and kept at 4 C overnight. Then 50 lL polymerized HRPanti mouse/rabbit lgG (KIT-9901, ElivisionTM plus Polymer HRP [mouse/rabbit] IHC Kit) was added, and nurtured at room temperature for 20 min, as specified by the manufacturer’s instructions. Freshly prepared diaminobenzidine (DAB) (50 lL) was required for each slide. Throughout the immunostaining process, except for the steps during which BSA and DAB were added, slides were washed in PBS three times. After immunostaining, the sections were stained with Mayer’s hematoxylin. Histomorphometric analysis For hard-tissue histology, the proximal parts of the left tibiae with implants were fixed in a 4% neutral formalinbuffered solution for 3 days, dehydrated with graded ethanol, and then embedded in methyl methacrylate for 30 days without decalcification. Horizontal cutting of sections (50 lm thick) were performed using a model microtome (SP1600, Leica Microsystems, Wetzlar, Germany). Sections approximately 4 mm below the epiphyseal plate were selected and stained in 1% toluidine blue and 5% acid fuchsin to observe bone-to-implant contact (BC) and bone area ratio (BA). BC was measured as the linear percentage of the interface with direct BC to total interface of the implant in the cancellous bone. BA was calculated as the area percentage of bone tissue to the whole area, which was defined as a ring extending 100 mm from the implant surface.39 Statistical analysis Each experiment was repeated three times. The assays were processed in triplicate, and the results are expressed as means 6 standard deviations. A one-way analysis of variance (ANOVA) was combined with a Student–Newman–Keuls (SNK) post-hoc test to determine statistical significance; p < 0.05 and p < 0.01 were considered statistically significant. RESULTS
Surface characterization Figure 1(A,B) show SEM pictures of the TiO2-NTs and the Ag-loaded TiO2-NTs samples. A highly ordered nanotube
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FIGURE 1. SEM images of the samples: (A) TiO2-NTs, (B) NT-Ag. XRD patterns of samples: (C) TiO2-Nts and NT-Ag. TEM image: (D) NT-Ag. AFM images of (E) TiO2-NTs, and (F) NT-Ag, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
array (inner diameter: 130–140 nm; length: 7 mm) was produced as a result of anodization and annealing in an air atmosphere. After soaking and irradiation, the nanotubes were coupled by a series of uniform nanoparticles in the inner wall. Figure 1(C) shows the X-ray diffraction (XRD) patterns of the specimens. The peaks observed from the TiO2-NTs were assigned to the anatase phase.15 As shown in Figure 1(D), the inner walls of the TiO2-NTs had a diameter of 10–20 nm. Finally, as shown in the AFM images in Figure 1(E,F), the surfaces of the TiO2-NTs and NT-Ag had similar roughness values of several hundred nanometers. Ag release The Ag release from the NT-Ag samples in PBS is depicted in Figure 2. The amount of Ag released diminished gradually and slowly with longer immersion times; however, a considerable amount remained after 2 weeks.
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Antibacterial assay and antiadhesive assay Figure 3(A–D) shows the Rap and Raa values. The TiO2-NTs samples (foils and rods) exhibited a constant Rap value of
FIGURE 2. Non-cumulative silver release profiles from NT-Ag into PBS.
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FIGURE 3. Picture (A) and (C) present antibacterial rates of the foil and rod samples to planktonic bacteria in the medium (Raa), and picture (B) and (D) show antibacterial rates of the foil and rod samples to adherent bacteria on the samples (Rap). The antibacterial assays data are expressed as means 6 standard deviations (n 5 3). One-way ANOVA test is used to determine the level of significance. p < 0.01.
30% over the 30-day experimental period. The NT-Ag samples (foils and rods) displayed a higher Rap value of 90%, without an obvious decrease until the end of the experiment. Ag was also effective at killing bacteria colonies on the NT-Ag samples over the 30-day period, as shown in Figure 3(B). The samples exhibited Raa values of nearly 90%; no significant decline was evident over the course of
the experiment. Compared to the NT-Ag specimens, the antibacterial effect of the TiO2-NT samples was not as obvious, reaching only 20–30%. The anti-adhesive effect of the sample surface was verified by SEM, as shown in Figure 4. No bacteria colonization was evident on the surface of the NTAg; insignificant bacteria colonization was apparent on the surface of the TiO2-NT samples. In contrast, a much larger
FIGURE 4. SEM images of the samples after 24 and 48 h of incubation in 1 mL of the bacteria suspension in TSB containing 1.5 3 108 cells under aerobic conditions. Quantity of colony-forming units adherent to the surface of the samples have significant difference, and a number of bacteria (black arrow), which shrink and even break down can be observed on the samples of NT-Ag, and it can also be found on the surface of TiO2-NTs samples. Black scale bar 5 1 lm.
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FIGURE 5. Representative images showing the inhibition zone of the samples after 24 h of incubation at 37 C under aerobic conditions on Mueller-Hinton (M-H) plates, on which 1.5 3 108 colony-forming units (CFUs)/mL were spread evenly. The diameter of the ZOI was 1.6 cm for the NT-Ag foil samples and the ZOI was 1.2 3 1.0 cm for the NT-Ag rod samples. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
amount of bacteria was observed on the surface of the Ti samples. In the ZOI tests (Fig. 5), the diameter of the ZOI was 1.6 cm for the NT-Ag foil samples and the ZOI was 1.2 3 1.0 cm for the NT-Ag rod samples. However, the TiO2-NT samples showed no evidence of a ZOI.
PET examination 18 F-FDG accumulation was reported in terms of the standardized uptake value (SUV), which was calculated as the radioactivity of the region of interest (ROI) divided by the relative injected dose, expressed per kilogram of body weight. The activity ratios between the operated and non operated sides were calculated and depicted as an SUV
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ratio. In Figure 6, group I (Ti-rod implant) and II (TiO2-NT rod implant) rats displayed enhanced uptake on the operated side at day 5 (SUV ratio 5 2.12 6 0.18, and 2.25 6 0.22, respectively) and day 10 (3.20 6 0.24 and 3.34 6 0.25, respectively); these values were much lower than those observed for group III (NT-Ag rod implant) (day 5: 1.11 6 010; day 10: SUV ratio 5 1.02 6 0.11). Statistical analysis clearly showed a significant difference (p < 0.01) in SUV ratios between group III and group I and II.
In vivo radiology In Figure 7, rats in group I (Ti-rod implant) and group II (TiO2-NT rod implant) presented no clear difference regarding the results of X-ray examination, with nine rats exhibiting
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rats presented evidence of osteomyelitis40 in the shape of an abundant neutrophilic exudate at 2 weeks. Chronic inflammatory cell infiltration emerged in large numbers after 3 weeks, and intramedullary necrosis, including abscess and osteonecrosis, appeared in the medulla of the metaphysis, with mild chronic inflammation 4 weeks postsurgery. The group II (TiO2-NT rod implant) rats showed a significant amount of neutrophilic exudates at 2 weeks; no significant difference was observed at 3 or 4 weeks, compared to the group I samples. The group III (NT-Ag rod implant) rats at 2 weeks displayed a small amount of neutrophilic exudate, which is a normal inflammatory response resulting from implantation of the metal rod; no neutrophilic exudate was observed at 3 or 4 weeks. The immunohistochemical results were in agreement with the presence of bacteria in situ. Bacteria were evident in both group I and II rats, as single organisms and in colonies. The bacteria were primarily located around the capillary loops and within the center of the inflammation and microabscesses. Histomorphometry Histological micrographs showed the details of the bone-toimplant interface and the peri-implant bone tissue [Fig. 9(A)]. The sections selected for observation exhibited satisfactory BC and peri-implant bone mass after the 8-weekk period of bone healing around the implants. The BC and BA ratio analyses [Fig. 9(B,C)] indicated no difference among the three groups, thus indicating that the NT-Ag rod implants did not disturb normal osseointegration. DISCUSSION FIGURE 6. The PET images for 18F-FDG A,B demonstrate the whole body tracer distribution in a rat and show an enhanced uptake mainly in the left tibia, and knee-joint of rats of group I (Ti-rod implant) and II (TiO2-NT rod implant) at day 5 and 10, which can not be observed in group III (NT-Ag rod implant). Furthermore, the images demonstrate an enhanced uptake in the urinary bladder and liver (due to the tracer excretion), and the abdominal cavity (due to abdominal infection caused by bloodstream infection from the infected tibia), and in the brain and shoulder joints (unspecific uptake), but also in the kidneys and urinary bladder (due to tracer excretion). Picture (C) demonstrates the difference of SUV ratio between group III and groups I, II. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the classic symptoms of osteomyelitis. Within 2 weeks after surgery, groups I and II presented with trabecular bone absorption and bone deformity. Aggravated bone absorption and a slight periosteal reaction was evident after one more week. An apparent periosteal reaction, soft tissue shadows, and new bone formation appeared in the 4th week. The group III (NT-Ag rod implant) rats showed no signs of infection over the course of the measurements. Statistical analysis confirmed the significant difference (p < 0.01) in the number of infected rats between group III and groups I and II. Histological analysis and immunohistochemistry The results of histological analysis and immunohistochemistry were shown in Figure 8. The group I (Ti-rod implant)
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Infections with bacteria such as Staphylococcus aureus (MRSA, ATCC43300) in the form of biofilm formation on implant surfaces have always been a common problem. Prevention of bacterial colonization and biofilm formation on surgically implanted materials has been studied extensively.2–4,12,13,15 For its antimicrobial properties, Ag has been extensively used in water recycling and sanitization and for the treatment of wound infections.41 Through a variety of pharmaceutical devices, including vascular and urinary catheters, endotracheal tubes, and implantable prostheses, Ag has been applied to reduce bacterial colonization.41 The proper reservoir form of active Ag, which offers optimal antibacterial properties and excellent biointegration, is still being actively pursued.42–44 Although antibacterial coatings have been researched extensively, it remains challenging to find and produce a coating with relatively long-lasting antibacterial effects both in vivo and in vitro, because the coatings have a tendency to degrade in the physiological environment. In addition, the method commonly used for loading antibiotics has a limited release capacity for most coatings, thus constraining long-term antibacterial effects. For example, a protein layer will quickly deposit on an implant loaded with covalently bonded antibiotics under physiological conditions; thus, it is highly questionable whether or not these antibiotics have the ability to
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FIGURE 7. Radiographic images of rats with the three kinds of rod implants at 2, 3, and 4 weeks postsurgery. Rats of group I (Ti-rod implant) and II (TiO2-NT rod implant) present no difference regarding to the results of X-ray examination, with nine rats show classic symptoms of osteomyelitis. Within 2 weeks after surgery, group I and group II presented with trabecular bone absorption (white arrow) and bone deformity (black arrow). Aggravated bone absorption (white arrow) and a slight periosteal reaction (red arrow) was evident after one more week. An apparent periosteal reaction (red arrow), soft tissue shadows (yellow arrow), and new bone formation appeared in the 4th week. The group III (NT-Ag rod implant) rats showed no signs of infection over the course of the measurements. Brown scale bar 5 1cm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
penetrate this protein layer. In contrast, our NT-Ag coating, fabricated on Ti implants by electrochemical anodization,45 allows for long-lasting antibacterial effects for the following reasons. First, the bacteria are unable to develop resistance against Ag, and its broad antibacterial spectrum bodes well for in vivo applications.46 Second, the high efficacy of Ag at low concentrations and the TiO2-NTs reduce biofilm formation. Third, the reservoir provided by the nanotubes can be regulated as needed; i.e., larger reservoirs produce longterm antibacterial effects, while smaller reservoirs minimize the likelihood of cytotoxicity if the tissue is sensitive to Ag. Fourth, the inorganic nature of NT-Ag prevents its decomposition; thus, stability can be expected. In our previous study,36 the NT-Ag surfaces eliminated 100% of both planktonic and adherent cells of methicillinsensitive S. aureus (MSSA, ATCC 6538). However, pathogens involved in implant infections are diverse, and bacteria in biofilms can resist the host immune responses and even antibiotics.47–50 The restricted activity of antibiotics against implant infections limits their clinical effectiveness. This is especially the case in infections involving antibiotic-resistant bacterial strains (e.g., MRSA strains), which have increasingly become a threat to healthcare environments and the
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health of the general public. In this study, we evaluated the antibacterial ability of NT-Ag surfaces toward MRSA (ATCC 43300). The NT-Ag surfaces demonstrated strong antibacterial properties throughout the entire experimental period. About 90% of all planktonic bacteria were killed. Ag was also effective for killing bacteria colonization on the NT-Ag samples over the 30-day evaluation period; approximately 90% of all adherent bacteria were also killed. Even the samples tested against 1 mL bacteria suspensions, containing 105 CFU mL21 in vivo in TSB, showed excellent antibacterial properties over the first 30 days. As such, a much longer period of antibacterial protection under normal conditions could be expected, conjugated with the host defense. Although previous studies23,51 reported good performance outcomes for their antibacterial coatings, ZOI tests could not confirm the results; this was attributed to the requirement of direct contact with the antibacterial coating or material, in contrast to the release of Ag ions. In the current study, ZOI tests clearly showed the antibacterial effect of the NTAg samples on high bacterial concentrations (1.5 3 109 CFU mL21), along with good penetration into the protein layer on Mueller–Hinton plates. SEM results showed an enhanced anti-adherent ability of the NT-Ag samples; few bacteria
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FIGURE 8. Histological and IHC analysis of the three group rats contaminating different implants at 2, 3, and 4 weeks post surgery. Consistent with the radiographic analysis, histological analysis (H&E staining) of group I (Ti-rod implant) and group II (TiO2-NT rod implant) present evidence of acute pyogenic infection in the shape of an abundant neutrophilic exudate (white arrow) at 2 weeks, with chronic inflammatory cell infiltration (black) emerge in large numbers after 3 weeks, and intramedullary necrosis including abscess and osteonecrosis (red arrow) demonstrated in the medulla of the metaphysis with mild chronic inflammation 4 weeks post surgery. In contrast, no group III (NT-Ag rod implant) at 2 weeks displayed a small amount of neutrophilic exudate, which is normal inflammatory response because of implantation of the metal rod, and no neutrophilic exudate can be observed at 3 and 4 weeks. IHC (immunohistochemical) analysis proved the existence the bacterial in group I and II at 2, 3, and 4 weeks, respectively, but no bacterial survival in group III. Yellow scale bar 5 1000 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
colonization units were observed on the NT-Ag surfaces, and some of the existing units were already dead or dying. In contrast, the bacteria adhered to the TiO2 surface. SEM results also indicated that the NT-Ag surface discouraged the bacteria from approaching the surface, thus minimizing bacterial biofilm formation. It has been reported that the initial bacterial adhesion to the surface of an implant is the first step in biofilm formation, and the only step that can be
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reversed during the formation process; biofilm is extremely difficult to remove once attached. Thus, it is generally accepted that prohibiting initial adhesion is the most effective and promising way to prevent biofilm accumulation on implants.15,51 The experimental results from the animal model demonstrated that the NT-Ag surfaces also possessed excellent antibacterial ability in vivo. In the early stages, PET
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centrations.20,21 In our previous study,36 NT-Ag surfaces had little impact on normal cells. Experimental evidence has indicated that biomaterials containing an appropriate amount of Ag are compatible with mammalian cells including osteoblasts,1,16,18,20,21 and it has been suggested that some Ag-containing coatings even enhance osteoblast proliferation,17,52 fibroblast attachment, and endothelial cell response.53 In the current study, after a 12-week period of bone healing around the implants, BA and BC analyses presented no differences among the three groups. These results should help ease concerns over the safety of Ag release. In summary, we demonstrated that NT-Ag successfully could eliminate MRSA (ATCC, 43300) in vitro, and inhibited bacterial adherence and biofilm formation on the surface. To prepare NT-Ag for clinical application, this form of Ag delivery should be tested on larger animals, such as rabbits or dogs. In addition, the synergistic effect of NT-Ag with antibiotics, reported in a previous study,54 should be examined closely, to possibly lessen the required antibiotic dosage for bacteria such as MSSA. CONCLUSION
FIGURE 9. Histomorphometric images of the proximal tibiae with implants approximately 4 mm below the epiphyseal plate 8 weeks after implantation, and results of the BA (B) and BC (C) in histomorphometry. Blue scale bar 5 100 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
examination clearly shown that group I (Ti-rod implant) and group II (TiO2-NT rod implant) rats at day 5 and 10 after surgery had significantly higher SUV ratios compared to group III (NT-Ag rod implant) (Fig. 6). Within 2 weeks after surgery, groups I and II rats had bone deformities and other signs of infection, whereas group III rats showed no problematic or abnormal growth in the early or late stages of the in vivo experiment. Ultimately, groups I and II also presented evidence of chronic osteomyelitis (e.g., chronic inflammation, intramedullary necroses, abscesses, and osteonecrosis), and had been colonized by bacteria, as single organisms and in colonies. This was not the case for group III rats. These results from both in vivo and in vitro experiments demonstrate that the Ag coating effectively prevented bacterial adherence and biofilm formation on the Ti alloy implants. Although high doses of Ag can induce cytotoxicity, Ag is generally believed to be cytocompatible and safe at low con-
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From a materials and application perspective, NT-Ag possess the ability to kill strong bacteria in vivo and in vitro, and the ability to reduce adherent bacteria, leading to less biofilm formation over a 30-day period. The amount of Ag nanoparticles can be regulated by adjusting the AgNO3 concentration and immersion time. The release rate of the Ag can be modified by controlling the length and size of the TiO2-NTs, and the space between the TiO2-NTs. Antibacterial ability and normal osseointegration activities exist simultaneously, thus minimizing safety concerns regarding the use of NT-Ag. Notably, the experimental conditions in this study were much more severe than those encountered in normal situations in vivo. Regardless, the materials exhibited excellent antibacterial ability. The NT-Ag are expected to be much more effective over a longer period of time in practical applications, in conjunction with host defense and antibiotics. In addition, the NT-Ag fabrication processes presented in this study are simple, economical, and applicable to industry, dentistry, and orthopedics. REFERENCES 1. Hardes J, Ahrens H, Gebert C, Streitbuerger A, Buerger H, Erren M, Gunsel A, Wedemeyer C, Saxler G, Winkelmann W, Gosheger G. Lack of toxicological side-effects in silver-coated megaprostheses in humans. Biomaterials 2007;28:2869–2875. 2. Schmalzried TP, Amstutz HC, Au MK, Dorey FJ. Etiology of deep sepsis in total hip arthroplasty. The significance of hematogenous and recurrent infections. Clin Orthop Relat Res 1992;280:200–207. 3. Donlan RM. Biofilms: Microbial life on surfaces. Emerg Infect Dis 2002;8:881–890. 4. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001;358:135–138. 5. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999;284:1318–1322. 6. Schwank S, Rajacic Z, Zimmerli W, Blaser J. Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics. Antimicrob Agents Chemother 1998;42:895–898. 7. Jones SM, Morgan M, Humphrey TJ, Lappin-Scott H. Effect of vancomycin and rifampicin on meticillin-resistant Staphylococcus aureus biofilms. Lancet 2001;357:40–41.
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8. Ellington JK, Reilly SS, Ramp WK, Smeltzer MS, Kellam JF, Hudson MC. Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts. Microb Pathogenesis 1999;26:317–323. 9. Hudson MC, Ramp WK, Nicholson NC, Williams AS, Nousiainen MT. Internalization of Staphylococcus aureus by cultured osteoblasts. Microb Pathogenesis 1995;19:409–419. 10. Jevon M, Guo C, Ma B, Mordan N, Nair SP, Harris M, Henderson B, Bentley G, Meghji S. Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts. Infect Immun 1999;67:2677–2681. 11. Reilly SS, Hudson MC, Kellam JF, Ramp WK. In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts. Bone 2000;26:63–70. 12. Green SA, Ripley MJ. Chronic osteomyelitis in pin tracks. J Bone Joint Surg Am 1984;66:1092–1098. 13. Monteiro DR, Gorup LF, Takamiya AS, Ruvollo-Filho AC, de Camargo ER, Barbosa DB. The growing importance of materials that prevent microbial adhesion: Antimicrobial effect of medical devices containing silver. Int J Antimicrob Agents 2009;34:103–110. 14. Monzon M, Oteiza C, Leiva J, Lamata M, Amorena B. Biofilm testing of Staphylococcus epidermidis clinical isolates: Low performance of vancomycin in relation to other antibiotics. Diagn Microb Infec Dis 2002;44:319–324. 15. Zhao L, Chu PK, Zhang Y, Wu Z. Antibacterial coatings on titanium implants. J Biomed Mater Res B 2009;91:470–480. 16. Alt V, Bechert T, Steinrucke P, Wagener M, Seidel P, Dingeldein E, Domann E, Schnettler R. An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement. Biomaterials 2004;25:4383–4391. 17. Bosetti M, Masse A, Tobin E, Cannas M. Silver coated materials for external fixation devices: In vitro biocompatibility and genotoxicity. Biomaterials 2002;23:887–892. 18. Chen W, Liu Y, Courtney HS, Bettenga M, Agrawal CM, Bumgardner JD, Ong JL. In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating. Biomaterials 2006;27:5512–5517. 19. Necula BS, Fratila-Apachitei LE, Zaat SA, Apachitei I, Duszczyk J. In vitro antibacterial activity of porous TiO2-Ag composite layers against methicillin-resistant Staphylococcus aureus. Acta Biomater 2009;5:3573–3580. 20. Agarwal A, Weis TL, Schurr MJ, Faith NG, Czuprynski CJ, McAnulty JF, Murphy CJ, Abbott NL. Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells. Biomaterials 2010; 31:680–690. 21. Ramstedt M, Ekstrand-Hammarstrom B, Shchukarev AV, Bucht A, Osterlund L, Welch M, Huck WT. Bacterial and mammalian cell response to poly(3-sulfopropyl methacrylate) brushes loaded with silver halide salts. Biomaterials 2009;30:1524–1531. 22. Russell AD, Hugo WB. Antimicrobial activity and action of silver. Prog Med Chem 1994;31:351–370. 23. Juan L, Zhimin Z, Anchun M, Lei L, Jingchao Z. Deposition of silver nanoparticles on titanium surface for antibacterial effect. Int J Nanomed 2010;5:261–267. 24. Crespo-Monteiro N, Destouches N, Bois L, Chassagneux F, Reynaud S, Fournel T. Reversible and irreversible laser microinscription on silver-containing mesoporous titania films. Adv Mater 2010;22:3166–3170. 25. Liu Y, Zheng Z, Zara JN, Hsu C, Soofer DE, Lee KS, Siu RK, Miller LS, Zhang X, Carpenter D, Wang C, Ting K, Soo C. The antimicrobial and osteoinductive properties of silver nanoparticle/poly (DLlactic-co-glycolic acid)-coated stainless steel. Biomaterials 2012; 33:8745–8756. 26. Zhao L, Mei S, Chu PK, Zhang Y, Wu Z. The influence of hierarchical hybrid micro/nano-textured titanium surface with titania nanotubes on osteoblast functions. Biomaterials 2010;31:5072–5082. 27. Zhao L, Mei S, Wang W, Chu PK, Wu Z, Zhang Y. The role of sterilization in the cytocompatibility of titania nanotubes. Biomaterials 2010;31:2055–2063. 28. Oh SH, Finones RR, Daraio C, Chen LH, Jin S. Growth of nanoscale hydroxyapatite using chemically treated titanium oxide nanotubes. Biomaterials 2005;26:4938–4943.
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29. Oh S, Daraio C, Chen LH, Pisanic TR, Finones RR, Jin S. Significantly accelerated osteoblast cell growth on aligned TiO2 nanotubes. J Biomed Mater Res A 2006;78:97–103. 30. Brammer KS, Oh S, Cobb CJ, Bjursten LM, van der Heyde H, Jin S. Improved bone-forming functionality on diameter-controlled TiO(2) nanotube surface. Acta Biomater 2009;5:3215–3223. 31. Das K, Bose S, Bandyopadhyay A. TiO2 nanotubes on Ti: Influence of nanoscale morphology on bone cell-materials interaction. J Biomed Mater Res A 2009;90:225–237. 32. Crawford GA, Chawla N, Das K, Bose S, Bandyopadhyay A. Microstructure and deformation behavior of biocompatible TiO2 nanotubes on titanium substrate. Acta Biomater 2007;3: 359–367. 33. Popat KC, Eltgroth M, Latempa TJ, Grimes CA, Desai TA. Decreased Staphylococcus epidermis adhesion and increased osteoblast functionality on antibiotic-loaded titania nanotubes. Biomaterials 2007;28:4880–4888. 34. Peng L, Mendelsohn AD, LaTempa TJ, Yoriya S, Grimes CA, Desai TA. Long-term small molecule and protein elution from TiO2 nanotubes. Nano Lett 2009;9:1932–1936. 35. Liu J, Sonshine DA, Shervani S, Hurt RH. Controlled release of biologically active silver from nanosilver surfaces. ACS Nano 2010;4:6903–6913. 36. Zhao L, Wang H, Huo K, Cui L, Zhang W, Ni H, Zhang Y, Wu Z, Chu PK. Antibacterial nano-structured titania coating incorporated with silver nanoparticles. Biomaterials 2011;32:5706–5716. 37. Humblot V, Yala JF, Thebault P, Boukerma K, Hequet A, Berjeaud JM, Pradier CM. The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers. Biomaterials 2009;30:3503–3512. 38. Monzon M, Garcia-Alvarez F, Lacleriga A, Gracia E, Leiva J, Oteiza C, Amorena B. A simple infection model using pre-colonized implants to reproduce rat chronic Staphylococcus aureus osteomyelitis and study antibiotic treatment. J Orthopaed Res 2001;19: 820–826. 39. Li Y, Li Q, Zhu S, Luo E, Li J, Feng G, Liao Y, Hu J. The effect of strontium-substituted hydroxyapatite coating on implant fixation in ovariectomized rats. Biomaterials 2010;31:9006–9014. 40. Li D, Gromov K, Soballe K, Puzas JE, O’Keefe RJ, Awad H, Drissi H, Schwarz EM. Quantitative mouse model of implant-associated osteomyelitis and the kinetics of microbial growth, osteolysis, and humoral immunity. J Orthopaed Res 2008;26:96–105. 41. Zheng Z, Yin W, Zara JN, Li W, Kwak J, Mamidi R, Lee M, Siu RK, Ngo R, Wang J, Carpenter D, Zhang X, Wu B, Ting K, Soo C. The use of BMP-2 coupled - Nanosilver-PLGA composite grafts to induce bone repair in grossly infected segmental defects. Biomaterials 2010;31:9293–9300. 42. Chua PH, Neoh KG, Kang ET, Wang W. Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion. Biomaterials 2008;29:1412–1421. 43. Zhang F, Zhang Z, Zhu X, Kang ET, Neoh KG. Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion. Biomaterials 2008;29:4751–4759. 44. Harris LG, Tosatti S, Wieland M, Textor M, Richards RG. Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-functionalized poly(L-lysine)grafted-poly(ethylene glycol) copolymers. Biomaterials 2004;25: 4135–4148. 45. Albu SP, Ghicov A, Macak JM, Hahn R, Schmuki P. Self-organized, free-standing TiO2 nanotube membrane for flow-through photocatalytic applications. Nano Lett 2007;7:1286–1289. 46. Percival SL, Bowler PG, Russell D. Bacterial resistance to silver in wound care. J Hosp Infect 2005;60:1–7. 47. Darouiche RO. Treatment of infections associated with surgical implants. New Engl J Med 2004;350:1422–1429. 48. Habash M, Reid G. Microbial biofilms: Their development and significance for medical device-related infections. J Clin Pharmacol 1999;39:887–898. 49. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, LappinScott HM. Microbial biofilms. Annu Rev Microbiol 1995;49:711– 745.
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50. Gristina AG. Implant failure and the immuno-incompetent fibroinflammatory zone. Clin Orthop Relat Res 1994;298:106–118. 51. Zheng Y, Li J, Liu X, Sun J. Antimicrobial and osteogenic effect of Ag-implanted titanium with a nanostructured surface. Int J Nanomed 2012;7:875–884. 52. Verne E, Di Nunzio S, Bosetti M, Appendino P, Brovarone CV, Maina G, Cannas M. Surface characterization of silver-doped bioactive glass. Biomaterials 2005;26:5111–5119.
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53. Hsu SH, Tseng HJ, Lin YC. The biocompatibility and antibacterial properties of waterborne polyurethane-silver nanocomposites. Biomaterials 2010;31:6796–6808. 54. Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: A study against gram-positive and gram-negative bacteria. Nanomed-Nanotechnol 2010;6:103– 109.
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Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China 2 School of Materials and Metallurgy, Wuhan University of Science and Technology, Wuhan 430081, China Received 9 August 2013; revised 5 October 2013; accepted 23 October 2013 Published online 15 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35019 Abstract: Although titanium (Ti) implants are widely used clinically, implant-associated bacterial infection is still one of the most serious complications in orthopedic surgery. Long-term antibacterial properties and the ability to inhibit biofilm formation are highly desirable to prevent implant associated infection. In this study, a controllable amount of silver (Ag) nanoparticles was incorporated into titanium oxide; or titanium, nanotubes (TiO2-NTs). The reliable release and long-term antibacterial function of Ag, in vivo and in vitro, and influence normal boneimplant integration from the Ag released from Ag-incorporated NTs in vivo have been studied to make them useable in clinical practice. In the current study, TiO2-NTs loaded with Ag (NT-Ag) exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) in vitro for 30 days,
and the ability to penetrate the protein layer well. In addition, Xray examination and 2-[18F]-fiuoro-2-deoxy-D-glucose positron emission tomography indicates that NT-Ag show extremely long antibacterial activity in vivo in a rat model. Furthermore, histomorphometric analysis demonstrated that satisfactory biointegration can be expected. Our results indicate that NT-Ag has both simultaneous antimicrobial and excellent bio-integration properties, make it a promising therapeutic material for orthopeC 2013 Wiley Periodicals, Inc. J Biomed Mater Res dic application. V Part A: 102A: 3488–3499, 2014.
Key Words: implant-associated infections, rat model, long antibacterial activity, silver nanoparticle, titania nanotubes (TiO2-NTs)
How to cite this article: Cheng H, Li Y, Huo K, Gao B, Xiong W. 2014. Long-lasting in vivo and in vitro antibacterial ability of nanostructured titania coating incorporated with silver nanoparticles. J Biomed Mater Res Part A 2014:102A:3488–3499.
INTRODUCTION
Postoperative infection associated with implants remains one of the most devastating complications in orthopedic surgery, sometimes demanding implant removal and/or repeated surgeries resulting in huge substantial costs and extended hospitalization. Usually these infections are difficult to diagnose in the early stages, which allows the invading bacteria to form a biofilm. This biofilm acts as a protective screen against antibacterial agents and host defense. In addition, the bacteria in a biofilm form will have a lower metabolic rate, making eradication, and detachment of the infective pathogens difficult, especially for growthdependent antibiotics.1–4 Antibiotic concentration can be up to 1000-fold higher needed to inhibit the growth of bacteria in biofilms compared to the planktonic bacteria.4,5–7 In addition, internalization of Staphylococcus aureus by osteoblast protects the bacteria from the host immune system,8–11 thus forming a reservoir of bacteria for recurring infection.12,13 By residing a longer time in vivo, the biofilms gradually become more resistant to antibiotics.14 Antibacterial coatings fabricated on the surfaces of biomedical devices can decrease the risk of the infection with
such bacteria by preventing microbe adhesion and proliferation. For such clinical applications, coatings must have combination of excellent antibacterial efficacy and low little toxicity to eukaryotic cells. Recently, significant attention has been given to antibacterial coatings containing silver (Ag), which has long been known to serve as an antibacterial agent that kills and inhibits a wide spectrum of microorganisms, including antibiotic-resistant bacteria, while possessing several other advantages such as satisfactory, stability, little possibility to develop antibiotic resistant strains, and non-cytotoxic at appropriate doses.1,15–21 In particular, Ag ions seems to show the best antibacterial activity.22–25 Although materials containing Ag have been widely explored for use in preventing biofilm formation, controlling the dosage and obtaining consistent therapeutic outcomes have been challenging. Titania nanotubes (TiO2-NTs) fabricated on titanium (Ti) implants by electrochemical anodization solves both of these issues.26–33 TiO2-NTs also offer the possibility of long-term antibacterial treatment time by controlling the release of nanoparticles such as Ag. For example, Ag release from TiO2-NTs can be regulated by the tube dimensions.34 The diameter of the TiO2-NTs and the space between nanotubes can be varied
Correspondence to: W. Xiong; e-mail: [email protected]
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from hundreds to tens of nanometers by adjusting several anodization parameters, thus, offering the most direct, feasible method for achieving the controlled release of Ag. Moreover, there is evidence that a range of competing chemical approaches can make the release of biologically active Ag controllable by changing the oxidation reactions in water.35 One of the other merits of this approach is that TiO2-NTs have a lower elastic modulus;32 thus, better biomechanical compatibility can be expected, compared to other artificial biomaterials, due to the close match with the elastic modulus of natural bone. TiO2-NTs induce mineralization, extracellular matrix secretion of nanostructured hydroxyapatite growing in vivo,28 and simultaneously promote osteogenic differentiation and mesenchymal stem cells proliferation.27,29–31 TiO2-NTs could act as carrier, which can store and release Ag ions in a controllable manner, showing long-term antibacterial capability. Generally speaking, TiO2-NTs are ideal bioactive coatings that can serve as carriers for Ag ions and other antibacterial agents, and simultaneously induce direct bone-implant bonding; however, clinical application additional in vivo studies for further verification. In this study, we investigate whether a proper quantity Ag incorporated into TiO2-NTs could be reliably released, and if the Ag loaded TiO2-NTs (NT-Ag) possesses long-term antibacterial function, both in vivo and in vitro. In addition, we studied the osseointegration property of NT-Ag in vivo. MATERIALS AND METHODS
Specimen preparation In brief, Ti foil (99.7% pure, Aldrich, 10 3 20 3 1 mm3) and titanium rods (diameter: 0.8 mm; length: 12 mm) were prepared. The TiO2-NTs were fabricated by electrochemical anodization, using a direct-current (DC) power supply (IT6834, ITECH, China). Ethylene glycol containing 0.5 wt% ammonium fluoride (NH4F) and 5 vol% distilled deionized (DI) water was used as an electrolyte. After anodization at 60 V for 1 h, amorphous TiO2-NTs (TiO2-NTAs) formed on the Ti foils. The amorphous form was converted into anatase TiO2-NTs by annealing at 450 C in air for 3 h. The anatase TiO2-NTs were soaked in AgNO3 solution (>99.8% purity) (H2O:C2H5OH 5 1:1) with a concentration of 1.5 M for 30 min. The samples were rinsed carefully with DI water and dried in an air stream. Then the samples were irradiated with ultraviolet light for 30 min using a highpressure Hg lamp. Surface characterization The surface morphology, topography, and structure of the TiO2-NTs and NT-Ag specimens were characterized by fieldemission scanning electron microscopy (FE-SEM, FEI Nova 400 Nano), transmission electron microscopy (TEM, Philips CM20), and atomic force microscopy (AFM, Auto-Probe CP, Park Scientific Instruments). Ag release The content of Ag released from the NT-Ag samples was monitored in phosphate-buffered saline (PBS) as follows.
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Firstly, the samples were immersed in 6 mL of PBS for 1 day in the dark and then taken out, and washed with DI water. The samples were dipped again in 6 mL of fresh PBS, and the process was repeated for a total 14 days to obtain the solution with released Ag1 content. The PBS solution containing released silver was analyzed by inductivelycoupled plasma-atomic emission spectrometry (ICP-AES, IRIS Advantage ER/S). Antibacterial assay Foil and rod samples were prepared as described earlier. The antibacterial activity was tested against methicillinresistant Staphylococcus aureus (MRSA, ATCC43300) cultivated in a trypticase soy broth (TSB) medium at 37 C for 18 h. It was diluted to a concentration of 105 CFU mL21 in the antibacterial assay. Each rod specimen and foil specimen was incubated in 1 mL of the bacteria suspension in TSB at 37 C for 1 day. The culture medium was sampled to determine the viable counts of planktonic bacteria. Non-adherent bacteria were eliminated by three gentle rinses with PBS. Then the each specimen was placed in 1 mL of TSB followed by ultrasonic vibration (40 W) for 5 min to detach the adhered bacteria. The resulting suspension was sampled to count the viable bacteria adhered to the specimens. The specimens were repeatedly used for a total incubation time of 30 days after ultrasonic cleaning, and sterilization by moist heat disinfection. The live bacteria in the sampled suspensions were counted using the serial dilution and the spread-plate method at days 1, 4, 7, 10, 15, 20, and 30. These sampling time points were chosen based on previous studies.36,37 The antibacterial effect on planktonic bacteria and adhered bacteria was represented by a bactericidal ratio, based on the following formulas: (1) the bactericidal ratio of planktonic bacteria growing in the TSB (Rap) (%) 5 (B2A) / A 3 100%, and (2) the bactericidal ratio of adhered bacteria on the surface of the specimens (Raa) (%) 5 (D2C) / C 3 100%, where A is the mean number of viable bacteria in the TSB medium inoculated with NT-Ag or TiO2-NTs, B is the mean number of viable bacteria in the TSB medium inoculated with pure titanium foils, C is the mean number of viable bacteria on the NT-Ag or TiO2-NT specimens, and D is the mean number of viable bacteria on the pure Ti foils. Zone of inhibition (ZOI) tests were performed in which 10 mL S. aureus, adjusted to 1.5 3 108 CFUs mL21, was spread evenly over Mueller–Hinton plates. The foil and rod samples were placed on the prepared plates separately. The plates were incubated at 37 C under anaerobic conditions for 24 h and photographed to record the results. Antiadhesive assay The antiadhesive effect of the surface of the samples was qualitatively tested by scanning electron microscopy (SEM; S-4800; Hitachi Tokyo, Japan). The samples were immersed 1 mL bacteria suspension in TSB, which contained 1.5 3 108 cells cultured for 24 h at 37 C. Non-adherent bacteria were removed by three gentle rinses with PBS. The samples were subsequently fixed with glutaraldehyde solution (2.5%
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concentration) for 2 h at 4 C. Then the samples were dehydrated in an ethanol series (30, 50, 70, 90, and 100%). After dehydration, an amyl acetate solution was used to replace the ethanol for 1 h at room temperature, and then the samples were critical-point dried. The adherent bacteria on resultant samples were coated with Au in a sputter coater and imaged using SEM. Animal surgery All surgeries in the in vivo experiment were performed under protocols approved by the Ethics Committee for Animal Experiments of Huazhong University of Science and Technology, Wu Han, China (permit number 10-07-02). The study used a total of 60 adult male SD rats (average weight: 150 g), supplied by the Laboratory Animal Center of Tongji Medical College of Huazhong University of Science and Technology. Thirty rats were equally divided into three groups and implanted with the three kinds of rod samples only for histomorphometry analysis: group I (Ti-rod implant), group II (TiO2-NT rod implant), and group III (NTAg rod implant). The other 30 rats, distributed into three groups, were injected with 2 lL bacteria and inserted with implants simultaneously. The rats were kept under specific pathogen free (SPF) conditions in our Laboratory Animal Center. The rats were anesthetized with 50 mg pentobarbital per kg body weight by intraperitoneal injection before surgery. Details regarding the surgical procedure are described in a previous report.38 After shaving and sterilizing the site with povidone iodine, an incision was made across the left knee. A sterilized syringe needle (external diameter: 0.8 mm) was used to drill a hole through the tibial plateau, from the proximal end to the distal end. After the medullary cavity was inoculated with 1 lL bacteria suspension in TSB (which contained 1.5 3 108 cells mL21), the three different types of rod samples were implanted into the medullary cavity. The other 30 rats were implanted with the three types of rod implants only. The burr hole was sealed with bone wax; skin openings were closed by sutures and sterilized with povidone iodine. PET examination For 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET), the 30 rats injected with 1 lL bacteria were injected with 93 MBq 18F-FDP through the caudal vein on day 5 and day 10 after surgery. Then the rats were anesthetized as described earlier, and 60 min after injection of the 18F-FDG, PET (Discovery LS PET/CT system, GE) was performed. In vivo radiology X-ray examination (Kodak, Directview, DR3000) was performed 2, 3, and 4 weeks after surgery to assess longitudinal osteolysis of the rats. Histological analysis and immunohistochemistry Three rats in each group were euthanized anesthesia, with an overdose of pentobarbital at 2, 3, and 4 weeks. The left tibia was removed and separated from the soft tissue, and
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subsequently fixed in 4% paraformaldehyde for 12 h. Then the specimens were incubated in ethylenediaminetetraacetic acid (EDTA) and embedded in paraffin. Sagittal sections were cut into sections (5 lm thick) and then were stained with hematoxylin and eosin (H&E). The sagittal sections were used for immunostaining. An indirect immunostaining technique, based on a specific S. aureus murine monoclonal antibody (ab37644; abcam plc, Cambridge, UK), was used for the in situ identification of S. aureus. Antigen retrieval was performed using a microwave repair treatment for 15 min after deparaffinization. Blocking of endogenous peroxidase activity was performed by placing the slides in 5% H2O2 for 10–30 min. After the addition of 5% bovine serum albumin (BSA), the reaction was developed for 30 min at room temperature. Then the specific S. aureus murine monoclonal antibody was added and kept at 4 C overnight. Then 50 lL polymerized HRPanti mouse/rabbit lgG (KIT-9901, ElivisionTM plus Polymer HRP [mouse/rabbit] IHC Kit) was added, and nurtured at room temperature for 20 min, as specified by the manufacturer’s instructions. Freshly prepared diaminobenzidine (DAB) (50 lL) was required for each slide. Throughout the immunostaining process, except for the steps during which BSA and DAB were added, slides were washed in PBS three times. After immunostaining, the sections were stained with Mayer’s hematoxylin. Histomorphometric analysis For hard-tissue histology, the proximal parts of the left tibiae with implants were fixed in a 4% neutral formalinbuffered solution for 3 days, dehydrated with graded ethanol, and then embedded in methyl methacrylate for 30 days without decalcification. Horizontal cutting of sections (50 lm thick) were performed using a model microtome (SP1600, Leica Microsystems, Wetzlar, Germany). Sections approximately 4 mm below the epiphyseal plate were selected and stained in 1% toluidine blue and 5% acid fuchsin to observe bone-to-implant contact (BC) and bone area ratio (BA). BC was measured as the linear percentage of the interface with direct BC to total interface of the implant in the cancellous bone. BA was calculated as the area percentage of bone tissue to the whole area, which was defined as a ring extending 100 mm from the implant surface.39 Statistical analysis Each experiment was repeated three times. The assays were processed in triplicate, and the results are expressed as means 6 standard deviations. A one-way analysis of variance (ANOVA) was combined with a Student–Newman–Keuls (SNK) post-hoc test to determine statistical significance; p < 0.05 and p < 0.01 were considered statistically significant. RESULTS
Surface characterization Figure 1(A,B) show SEM pictures of the TiO2-NTs and the Ag-loaded TiO2-NTs samples. A highly ordered nanotube
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FIGURE 1. SEM images of the samples: (A) TiO2-NTs, (B) NT-Ag. XRD patterns of samples: (C) TiO2-Nts and NT-Ag. TEM image: (D) NT-Ag. AFM images of (E) TiO2-NTs, and (F) NT-Ag, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
array (inner diameter: 130–140 nm; length: 7 mm) was produced as a result of anodization and annealing in an air atmosphere. After soaking and irradiation, the nanotubes were coupled by a series of uniform nanoparticles in the inner wall. Figure 1(C) shows the X-ray diffraction (XRD) patterns of the specimens. The peaks observed from the TiO2-NTs were assigned to the anatase phase.15 As shown in Figure 1(D), the inner walls of the TiO2-NTs had a diameter of 10–20 nm. Finally, as shown in the AFM images in Figure 1(E,F), the surfaces of the TiO2-NTs and NT-Ag had similar roughness values of several hundred nanometers. Ag release The Ag release from the NT-Ag samples in PBS is depicted in Figure 2. The amount of Ag released diminished gradually and slowly with longer immersion times; however, a considerable amount remained after 2 weeks.
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Antibacterial assay and antiadhesive assay Figure 3(A–D) shows the Rap and Raa values. The TiO2-NTs samples (foils and rods) exhibited a constant Rap value of
FIGURE 2. Non-cumulative silver release profiles from NT-Ag into PBS.
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FIGURE 3. Picture (A) and (C) present antibacterial rates of the foil and rod samples to planktonic bacteria in the medium (Raa), and picture (B) and (D) show antibacterial rates of the foil and rod samples to adherent bacteria on the samples (Rap). The antibacterial assays data are expressed as means 6 standard deviations (n 5 3). One-way ANOVA test is used to determine the level of significance. p < 0.01.
30% over the 30-day experimental period. The NT-Ag samples (foils and rods) displayed a higher Rap value of 90%, without an obvious decrease until the end of the experiment. Ag was also effective at killing bacteria colonies on the NT-Ag samples over the 30-day period, as shown in Figure 3(B). The samples exhibited Raa values of nearly 90%; no significant decline was evident over the course of
the experiment. Compared to the NT-Ag specimens, the antibacterial effect of the TiO2-NT samples was not as obvious, reaching only 20–30%. The anti-adhesive effect of the sample surface was verified by SEM, as shown in Figure 4. No bacteria colonization was evident on the surface of the NTAg; insignificant bacteria colonization was apparent on the surface of the TiO2-NT samples. In contrast, a much larger
FIGURE 4. SEM images of the samples after 24 and 48 h of incubation in 1 mL of the bacteria suspension in TSB containing 1.5 3 108 cells under aerobic conditions. Quantity of colony-forming units adherent to the surface of the samples have significant difference, and a number of bacteria (black arrow), which shrink and even break down can be observed on the samples of NT-Ag, and it can also be found on the surface of TiO2-NTs samples. Black scale bar 5 1 lm.
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FIGURE 5. Representative images showing the inhibition zone of the samples after 24 h of incubation at 37 C under aerobic conditions on Mueller-Hinton (M-H) plates, on which 1.5 3 108 colony-forming units (CFUs)/mL were spread evenly. The diameter of the ZOI was 1.6 cm for the NT-Ag foil samples and the ZOI was 1.2 3 1.0 cm for the NT-Ag rod samples. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
amount of bacteria was observed on the surface of the Ti samples. In the ZOI tests (Fig. 5), the diameter of the ZOI was 1.6 cm for the NT-Ag foil samples and the ZOI was 1.2 3 1.0 cm for the NT-Ag rod samples. However, the TiO2-NT samples showed no evidence of a ZOI.
PET examination 18 F-FDG accumulation was reported in terms of the standardized uptake value (SUV), which was calculated as the radioactivity of the region of interest (ROI) divided by the relative injected dose, expressed per kilogram of body weight. The activity ratios between the operated and non operated sides were calculated and depicted as an SUV
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ratio. In Figure 6, group I (Ti-rod implant) and II (TiO2-NT rod implant) rats displayed enhanced uptake on the operated side at day 5 (SUV ratio 5 2.12 6 0.18, and 2.25 6 0.22, respectively) and day 10 (3.20 6 0.24 and 3.34 6 0.25, respectively); these values were much lower than those observed for group III (NT-Ag rod implant) (day 5: 1.11 6 010; day 10: SUV ratio 5 1.02 6 0.11). Statistical analysis clearly showed a significant difference (p < 0.01) in SUV ratios between group III and group I and II.
In vivo radiology In Figure 7, rats in group I (Ti-rod implant) and group II (TiO2-NT rod implant) presented no clear difference regarding the results of X-ray examination, with nine rats exhibiting
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rats presented evidence of osteomyelitis40 in the shape of an abundant neutrophilic exudate at 2 weeks. Chronic inflammatory cell infiltration emerged in large numbers after 3 weeks, and intramedullary necrosis, including abscess and osteonecrosis, appeared in the medulla of the metaphysis, with mild chronic inflammation 4 weeks postsurgery. The group II (TiO2-NT rod implant) rats showed a significant amount of neutrophilic exudates at 2 weeks; no significant difference was observed at 3 or 4 weeks, compared to the group I samples. The group III (NT-Ag rod implant) rats at 2 weeks displayed a small amount of neutrophilic exudate, which is a normal inflammatory response resulting from implantation of the metal rod; no neutrophilic exudate was observed at 3 or 4 weeks. The immunohistochemical results were in agreement with the presence of bacteria in situ. Bacteria were evident in both group I and II rats, as single organisms and in colonies. The bacteria were primarily located around the capillary loops and within the center of the inflammation and microabscesses. Histomorphometry Histological micrographs showed the details of the bone-toimplant interface and the peri-implant bone tissue [Fig. 9(A)]. The sections selected for observation exhibited satisfactory BC and peri-implant bone mass after the 8-weekk period of bone healing around the implants. The BC and BA ratio analyses [Fig. 9(B,C)] indicated no difference among the three groups, thus indicating that the NT-Ag rod implants did not disturb normal osseointegration. DISCUSSION FIGURE 6. The PET images for 18F-FDG A,B demonstrate the whole body tracer distribution in a rat and show an enhanced uptake mainly in the left tibia, and knee-joint of rats of group I (Ti-rod implant) and II (TiO2-NT rod implant) at day 5 and 10, which can not be observed in group III (NT-Ag rod implant). Furthermore, the images demonstrate an enhanced uptake in the urinary bladder and liver (due to the tracer excretion), and the abdominal cavity (due to abdominal infection caused by bloodstream infection from the infected tibia), and in the brain and shoulder joints (unspecific uptake), but also in the kidneys and urinary bladder (due to tracer excretion). Picture (C) demonstrates the difference of SUV ratio between group III and groups I, II. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the classic symptoms of osteomyelitis. Within 2 weeks after surgery, groups I and II presented with trabecular bone absorption and bone deformity. Aggravated bone absorption and a slight periosteal reaction was evident after one more week. An apparent periosteal reaction, soft tissue shadows, and new bone formation appeared in the 4th week. The group III (NT-Ag rod implant) rats showed no signs of infection over the course of the measurements. Statistical analysis confirmed the significant difference (p < 0.01) in the number of infected rats between group III and groups I and II. Histological analysis and immunohistochemistry The results of histological analysis and immunohistochemistry were shown in Figure 8. The group I (Ti-rod implant)
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Infections with bacteria such as Staphylococcus aureus (MRSA, ATCC43300) in the form of biofilm formation on implant surfaces have always been a common problem. Prevention of bacterial colonization and biofilm formation on surgically implanted materials has been studied extensively.2–4,12,13,15 For its antimicrobial properties, Ag has been extensively used in water recycling and sanitization and for the treatment of wound infections.41 Through a variety of pharmaceutical devices, including vascular and urinary catheters, endotracheal tubes, and implantable prostheses, Ag has been applied to reduce bacterial colonization.41 The proper reservoir form of active Ag, which offers optimal antibacterial properties and excellent biointegration, is still being actively pursued.42–44 Although antibacterial coatings have been researched extensively, it remains challenging to find and produce a coating with relatively long-lasting antibacterial effects both in vivo and in vitro, because the coatings have a tendency to degrade in the physiological environment. In addition, the method commonly used for loading antibiotics has a limited release capacity for most coatings, thus constraining long-term antibacterial effects. For example, a protein layer will quickly deposit on an implant loaded with covalently bonded antibiotics under physiological conditions; thus, it is highly questionable whether or not these antibiotics have the ability to
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FIGURE 7. Radiographic images of rats with the three kinds of rod implants at 2, 3, and 4 weeks postsurgery. Rats of group I (Ti-rod implant) and II (TiO2-NT rod implant) present no difference regarding to the results of X-ray examination, with nine rats show classic symptoms of osteomyelitis. Within 2 weeks after surgery, group I and group II presented with trabecular bone absorption (white arrow) and bone deformity (black arrow). Aggravated bone absorption (white arrow) and a slight periosteal reaction (red arrow) was evident after one more week. An apparent periosteal reaction (red arrow), soft tissue shadows (yellow arrow), and new bone formation appeared in the 4th week. The group III (NT-Ag rod implant) rats showed no signs of infection over the course of the measurements. Brown scale bar 5 1cm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
penetrate this protein layer. In contrast, our NT-Ag coating, fabricated on Ti implants by electrochemical anodization,45 allows for long-lasting antibacterial effects for the following reasons. First, the bacteria are unable to develop resistance against Ag, and its broad antibacterial spectrum bodes well for in vivo applications.46 Second, the high efficacy of Ag at low concentrations and the TiO2-NTs reduce biofilm formation. Third, the reservoir provided by the nanotubes can be regulated as needed; i.e., larger reservoirs produce longterm antibacterial effects, while smaller reservoirs minimize the likelihood of cytotoxicity if the tissue is sensitive to Ag. Fourth, the inorganic nature of NT-Ag prevents its decomposition; thus, stability can be expected. In our previous study,36 the NT-Ag surfaces eliminated 100% of both planktonic and adherent cells of methicillinsensitive S. aureus (MSSA, ATCC 6538). However, pathogens involved in implant infections are diverse, and bacteria in biofilms can resist the host immune responses and even antibiotics.47–50 The restricted activity of antibiotics against implant infections limits their clinical effectiveness. This is especially the case in infections involving antibiotic-resistant bacterial strains (e.g., MRSA strains), which have increasingly become a threat to healthcare environments and the
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health of the general public. In this study, we evaluated the antibacterial ability of NT-Ag surfaces toward MRSA (ATCC 43300). The NT-Ag surfaces demonstrated strong antibacterial properties throughout the entire experimental period. About 90% of all planktonic bacteria were killed. Ag was also effective for killing bacteria colonization on the NT-Ag samples over the 30-day evaluation period; approximately 90% of all adherent bacteria were also killed. Even the samples tested against 1 mL bacteria suspensions, containing 105 CFU mL21 in vivo in TSB, showed excellent antibacterial properties over the first 30 days. As such, a much longer period of antibacterial protection under normal conditions could be expected, conjugated with the host defense. Although previous studies23,51 reported good performance outcomes for their antibacterial coatings, ZOI tests could not confirm the results; this was attributed to the requirement of direct contact with the antibacterial coating or material, in contrast to the release of Ag ions. In the current study, ZOI tests clearly showed the antibacterial effect of the NTAg samples on high bacterial concentrations (1.5 3 109 CFU mL21), along with good penetration into the protein layer on Mueller–Hinton plates. SEM results showed an enhanced anti-adherent ability of the NT-Ag samples; few bacteria
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FIGURE 8. Histological and IHC analysis of the three group rats contaminating different implants at 2, 3, and 4 weeks post surgery. Consistent with the radiographic analysis, histological analysis (H&E staining) of group I (Ti-rod implant) and group II (TiO2-NT rod implant) present evidence of acute pyogenic infection in the shape of an abundant neutrophilic exudate (white arrow) at 2 weeks, with chronic inflammatory cell infiltration (black) emerge in large numbers after 3 weeks, and intramedullary necrosis including abscess and osteonecrosis (red arrow) demonstrated in the medulla of the metaphysis with mild chronic inflammation 4 weeks post surgery. In contrast, no group III (NT-Ag rod implant) at 2 weeks displayed a small amount of neutrophilic exudate, which is normal inflammatory response because of implantation of the metal rod, and no neutrophilic exudate can be observed at 3 and 4 weeks. IHC (immunohistochemical) analysis proved the existence the bacterial in group I and II at 2, 3, and 4 weeks, respectively, but no bacterial survival in group III. Yellow scale bar 5 1000 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
colonization units were observed on the NT-Ag surfaces, and some of the existing units were already dead or dying. In contrast, the bacteria adhered to the TiO2 surface. SEM results also indicated that the NT-Ag surface discouraged the bacteria from approaching the surface, thus minimizing bacterial biofilm formation. It has been reported that the initial bacterial adhesion to the surface of an implant is the first step in biofilm formation, and the only step that can be
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reversed during the formation process; biofilm is extremely difficult to remove once attached. Thus, it is generally accepted that prohibiting initial adhesion is the most effective and promising way to prevent biofilm accumulation on implants.15,51 The experimental results from the animal model demonstrated that the NT-Ag surfaces also possessed excellent antibacterial ability in vivo. In the early stages, PET
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centrations.20,21 In our previous study,36 NT-Ag surfaces had little impact on normal cells. Experimental evidence has indicated that biomaterials containing an appropriate amount of Ag are compatible with mammalian cells including osteoblasts,1,16,18,20,21 and it has been suggested that some Ag-containing coatings even enhance osteoblast proliferation,17,52 fibroblast attachment, and endothelial cell response.53 In the current study, after a 12-week period of bone healing around the implants, BA and BC analyses presented no differences among the three groups. These results should help ease concerns over the safety of Ag release. In summary, we demonstrated that NT-Ag successfully could eliminate MRSA (ATCC, 43300) in vitro, and inhibited bacterial adherence and biofilm formation on the surface. To prepare NT-Ag for clinical application, this form of Ag delivery should be tested on larger animals, such as rabbits or dogs. In addition, the synergistic effect of NT-Ag with antibiotics, reported in a previous study,54 should be examined closely, to possibly lessen the required antibiotic dosage for bacteria such as MSSA. CONCLUSION
FIGURE 9. Histomorphometric images of the proximal tibiae with implants approximately 4 mm below the epiphyseal plate 8 weeks after implantation, and results of the BA (B) and BC (C) in histomorphometry. Blue scale bar 5 100 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
examination clearly shown that group I (Ti-rod implant) and group II (TiO2-NT rod implant) rats at day 5 and 10 after surgery had significantly higher SUV ratios compared to group III (NT-Ag rod implant) (Fig. 6). Within 2 weeks after surgery, groups I and II rats had bone deformities and other signs of infection, whereas group III rats showed no problematic or abnormal growth in the early or late stages of the in vivo experiment. Ultimately, groups I and II also presented evidence of chronic osteomyelitis (e.g., chronic inflammation, intramedullary necroses, abscesses, and osteonecrosis), and had been colonized by bacteria, as single organisms and in colonies. This was not the case for group III rats. These results from both in vivo and in vitro experiments demonstrate that the Ag coating effectively prevented bacterial adherence and biofilm formation on the Ti alloy implants. Although high doses of Ag can induce cytotoxicity, Ag is generally believed to be cytocompatible and safe at low con-
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From a materials and application perspective, NT-Ag possess the ability to kill strong bacteria in vivo and in vitro, and the ability to reduce adherent bacteria, leading to less biofilm formation over a 30-day period. The amount of Ag nanoparticles can be regulated by adjusting the AgNO3 concentration and immersion time. The release rate of the Ag can be modified by controlling the length and size of the TiO2-NTs, and the space between the TiO2-NTs. Antibacterial ability and normal osseointegration activities exist simultaneously, thus minimizing safety concerns regarding the use of NT-Ag. Notably, the experimental conditions in this study were much more severe than those encountered in normal situations in vivo. Regardless, the materials exhibited excellent antibacterial ability. The NT-Ag are expected to be much more effective over a longer period of time in practical applications, in conjunction with host defense and antibiotics. In addition, the NT-Ag fabrication processes presented in this study are simple, economical, and applicable to industry, dentistry, and orthopedics. REFERENCES 1. Hardes J, Ahrens H, Gebert C, Streitbuerger A, Buerger H, Erren M, Gunsel A, Wedemeyer C, Saxler G, Winkelmann W, Gosheger G. Lack of toxicological side-effects in silver-coated megaprostheses in humans. Biomaterials 2007;28:2869–2875. 2. Schmalzried TP, Amstutz HC, Au MK, Dorey FJ. Etiology of deep sepsis in total hip arthroplasty. The significance of hematogenous and recurrent infections. Clin Orthop Relat Res 1992;280:200–207. 3. Donlan RM. Biofilms: Microbial life on surfaces. Emerg Infect Dis 2002;8:881–890. 4. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001;358:135–138. 5. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999;284:1318–1322. 6. Schwank S, Rajacic Z, Zimmerli W, Blaser J. Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics. Antimicrob Agents Chemother 1998;42:895–898. 7. Jones SM, Morgan M, Humphrey TJ, Lappin-Scott H. Effect of vancomycin and rifampicin on meticillin-resistant Staphylococcus aureus biofilms. Lancet 2001;357:40–41.
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