Both Enhanced Biocompatibility and Antibacterial Activity in Ag-Decorated TiO2 Nanotubes

In this study, Ag is electron-beam evaporated to modify the topography of anodic TiO2 nanotubes of different diameters to obtain an implant with enhanced antibacterial activity and biocompatibility. We found that highly hydrophilic as-grown TiO2 nanotubes became poorly hydrophilic with Ag incorporation; however they could effectively recover their wettability to some extent under ultraviolet light irradiation. The results obtained from antibacterial tests suggested that the Ag-decorated TiO2 nanotubes could greatly inhibit the growth of Staphylococcus aureus. In vitro biocompatibility evaluation indicated that fibroblast cells exhibited an obvious diameter-dependent behavior on both as-grown and Ag-decorated TiO2 nanotubes. Most importantly, of all samples, the smallest diameter (25-nm-diameter) Ag-decorated nanotubes exhibited the most obvious biological activity in promoting adhesion and proliferation of human fibroblasts, and this activity could be attributed to the highly irregular topography on a nanometric scale of the Ag-decorated nanotube surface. These experimental results demonstrate that by properly controlling the structural parameters of Ag-decorated TiO2 nanotubes, an implant surface can be produced that enhances biocompatibility and simultaneously boosts antibacterial activity.


Introduction
Titanium (Ti) based alloys have been widely used to fabricate implantable devices such as artificial blood vessels, and orthopedic and dental implants because of their favorable mechanical properties, corrosion resistance, and biocompatibility [1][2][3]. When exposed to air, Ti forms a titanium oxide (TiO 2 ) layer on its surface (approximately 10 nm thick) acting like a ceramic with excellent biocompatibility. Once the Ti implant is inserted into the human body, the host tissues directly come into contact with the TiO 2 layer on the implant surface. Therefore, the surface characteristics of the TiO 2 layer dominate the biocompatibility of Ti-based implants. Recently, the interaction of nanometric scale surface topography with cells has been considered to be an increasingly important factor for tissue acceptance and cell functions [4][5][6]. Various nano-topographical modifications have been proposed to improve the cell responses to Ti-based implants. For example, TiO 2 nanowire scaffolds fabricated by the hydrothermal reaction of alkali with Ti metal, mimicking the natural extracellular matrix in structure, have been found to enhance the adhesion and proliferation of mesenchymal stem cells (MSCs) on Ti implants [7]. In addition, highly ordered TiO 2 nanotubes fabricated on Ti implants by electrochemical anodization have attracted considerable attention. A major advantage of anodic oxidation is the ability to precisely control the diameter and shape of the nanotubular arrays to the desired scale, meeting the demands of a specific application by precisely controlling the anodization parameters. In a number of studies on the cell responses to TiO 2 nanotubes, nanosize effects have been shown for a variety of cells [8][9][10][11]. Park et al. reported that vitality, proliferation, migration, and differentiation of MSCs and hematopoietic stem cells, as well as the behavior of osteoblasts and osteoclasts were strongly affected by the nanometric scale TiO 2 surface topography with a specific response to nanotube diameters between 15 and 100 nm [12]. Our recent study also reported the diameter-sensitive cytocompatibility of TiO 2 nanotubes treated with supercritical CO 2 fluid [13]. In other words, cell vitality has an extremely close relationship with the geometric factors of TiO 2 nanotube openings.
Host tissue characteristics are also crucial for the long-term success of inserted implants. When the implant itself damages or invades epithelial or mucosa barriers, it may serve as a reservoir for microorganisms thereby predisposing to infection. Once infection occurs, bacteria tend to aggregate in a hydrated polymeric matrix to form a bio-film on the implant which is difficult for the host defense and antimicrobial therapy to destroy [14,15]. Such implant-related infections may lead to removal of the implant, revision surgery and even amputation, all of which are associated with extremely high medical costs. It is generally accepted that the most effective method to prevent bio-film buildup on implants is to prohibit initial bacterial adhesion by making the implants antibacterial [16,17]. A variety of chemical forms of silver (Ag) have been widely used as antibacterial agents because of their strong broad-spectrum antibacterial activity, noncytotoxicity at suitable doses, and satisfactory stability [18][19][20]. It is believed that Ag in aqueous solution can release Ag ions which interact with the main components of bacterial cells such as DNA and proteins to cause the death of the bacterium [21,22]. Therefore, incorporating metallic Ag into Ti-based implants provides a feasible scheme for antibacterial bio-implants.
Recently, some research groups have attempted to develop Agloaded TiO 2 nanotubes as bio-implants from the viewpoint of combining enhanced biocompatibility and antibacterial activity. Das et al. reported that TiO 2 nanotubes electrodeposited with Ag had an antibacterial activity over 99% against the growth of colonies of Pseudomonas aeruginosa [23]. Recently, Zhao also reported that Ag incorporated into TiO 2 nanotubes by AgNO 3 immersion and ultraviolet (UV) irradiation possessed the capability to prevent bacterial adhesion without obvious decline for 30 days [24]. In both of these studies, all of the TiO 2 nanotubes had a diameter of approximately 100 nm and retained the original nanotubular structure with Ag nanoparticles incorporated into the nanotubes. Nevertheless, these Ag-loaded TiO 2 nanotubes still showed some cytotoxicity compared to Ag-free samples. Since many studies have shown that a low concentration of released Ag ions does not cause cytotoxicity [25,26], we hypothesized that by properly controlling the structural parameters of Ag-loaded TiO 2 nanotubes, an implant surface could be produced that enhances biocompatibility and simultaneously boosts antibacterial activity. In this study, Ag is electron-beam evaporated to modify the topography of anodic TiO 2 nanotubes with diameters ranging from 25 nm to 100 nm. We found that 25-nm-diameter TiO 2 nanotubes coated with 10-nm-thickness Ag not only exhibited enhanced antibacterial activity against Staphylococcus aureus (S. aureus), but also better cell responses to human fibroblasts. In addition, this activity could be attributed to the highly irregular topography on a nanometric scale of the surface of the Ag-coated nanotubes.

Materials and Methods
Preparation of Ag-decorated TiO 2 Nanotubes Self-organized TiO 2 nanotubes were fabricated by electrochemical anodization of Ti foil (thickness of 0.127 mm, 99.7% purity, ECHO Chemical Co. Ltd., Miaoli, Taiwan). A twoelectrode electrochemical cell with Ti as the anode and platinum (Pt) as the counter electrode was used. All anodization experiments were performed in ethylene glycol electrolytes containing 0.5 wt% NH 4 F at 20uC for 90 min, and all electrolytes were prepared from reagent-grade chemicals and deionized water. Anodization voltages were adjusted to result in TiO 2 nanotubes with diameters of 25, 50, and 100 nm. Subsequently, a 10-nm-thick Ag layer was electron-beam evaporated on these nanotubes. During the deposition of Ag, the vacuum level and deposition rate were maintained at 2610 7 Torr and 0.1 nm/s, respectively. For the invitro experiments, low-intensity UV light irradiation (,2 mW/ cm 2 ) was used on all nanotube samples using fluorescent blacklight bulbs for 8 h.

Material Characterization
Field emission scanning electron microscopy (FE-SEM; FEI Quanta 200 F, FEI, Hillsboro, OR, USA) was employed to characterize the surface morphology of the Ag-decorated nanotubes. X-ray diffraction (XRD; D2 Phaser, Bruker, Billerica, MA, USA) and transmission electron microscopy (TEM; JEM-2100, JEOL, Japan) in conjunction with an energy dispersion spectrometer (EDS) were utilized to determine the TiO 2 crystalline structure and Ag distribution in the nanotubes. The surface wettability of the materials was evaluated by measuring the contact angle between the TiO 2 nanotubes and water droplets in the dark. Contact angle measurements were performed at room temperature by the extension method using a horizontal microscope with a protractor eyepiece.

Ag Ion Release
The amount of Ag released from the Ag-decorated TiO 2 nanotubes was monitored in phosphate buffered saline (PBS) at 37uC. The Ag-decorated nanotubes were immersed in 10 ml of PBS for 1 day in the dark, taken out, and then immersed again in 10 ml of fresh PBS. This process was repeated for a total of 14 days to generate solutions at different time points in order to obtain the Ag release time profile. The PBS solution containing the released Ag ions was analyzed by inductively-coupled plasma mass spectrometry (ICP-MS; ELAN 6100, Perkin-Elmer, Waltham, MA, USA). The accumulative amounts of Ag ion release were presented in this study.  Human Fibroblast Cell Culture MRC-5 human fibroblasts were purchased from the Bioresource Collection and Research Center, Taiwan. The cells were plated in a 10-cm tissue culture plate and cultured with Eagle's minimum essential medium (Gibco, Life Technologies Corporation, Grand Island, NY, USA) containing 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate. Cultures were maintained at 37uC in a humidified atmosphere of 5% CO 2 . The cells were then seeded onto the autoclaved nanotube samples placed on the bottom of 12-well culture plate (Falcon, BD Biosciences, San Jose, CA, USA) at a density of 1610 4 cells/cm 2 for 3 days for cell adhesion and proliferation assay.

Cell Proliferation Assay
Cell viability was determined using a WST-1 cell proliferation reagent kit (Roche, Woerden, Netherlands) according to the manufacturer's instructions. On the 3rd day, cells on the   nanotubes were washed with PBS twice, and then incubated with a medium containing 10% WST-1 cell proliferation reagent at 37uC in a humidified atmosphere of 5% CO 2 for 2 h. The solution was then retrieved from each well and plated in a 96-well plate, and optical densities were measured using a spectrophotometer (Tecan Group Ltd., Mä nnedorf, Switzerland) at 450 nm.

Protein Adsorption
To evaluate the adsorption of proteins on the Ag-decorated TiO 2 nanotubes, bovine serum albumin (BSA; Sigma-Aldrich Corporation, St. Louis, MO, USA) was used as a model protein.
X-ray photoelectron spectroscopy (XPS) was employed to analyze the nitrogen spectra (in terms of N 1 s) on the nanotube surfaces after 5 min of immersion in BSA solution (5 mg BSA in 1 ml PBS).

Antibacterial Test
The antibacterial activity against S. aureus (ATCC 25923, Bioresource Collection and Research Center, Hsinchu, Taiwan) was then evaluated. S. aureus was cultured in tryptic soy broth and agar plates. The nanotube samples were sterilized in an autoclave at 121uC for 40 min, and then a suspension containing the bacteria at a concentration of 4610 9 cfu ml 21 was introduced onto the surface of the samples to a density of 150 ml cm 22 . The samples with the bacterial suspension were incubated at 37uC for 4 h. Subsequently, the samples were rinsed with sterile water and bacteria were detached by sonication from the surface of the nanotubes. The bacteria solution was collected and diluted 100X before being plated on an agar plate. After incubation at 37uC for 24 h, the active bacteria were counted and the agar plates were photographed. The examination of the morphology of the bacteria adhering to the nanotube surface was performed by SEM. The samples were prepared by plating a 2610 10

Statistical Analysis
All experiments were carried out in triplicate and at least three independent experiments were performed. The test values were expressed as mean 6 standard error (SE). In Ag ion release experiment, Student t test was used to compare Ag ion concentration between 25-and 100-nm-diameter groups. In cell proliferation assay, statistic comparisons of multi-group data were analyzed by ANOVA, followed by Scheffe's post-test using SPSS 12.0 software (SPSS Inc., Chicago, IL). A p value of less than 0.05 was considered to be statistically significant. Figure 1A-C shows the SEM images of the as-anodized TiO 2 nanotubes with diameters of 25 nm, 50 nm, and 100 nm, produced by electrochemical anodization at the applied voltages of 10 V, 20 V, and 40 V, respectively. These as-grown TiO 2 nanotubes had well-defined nanotubular structure, and their nanotube diameters were nearly proportional to the applied voltages. In addition, the XRD results from our previous study confirmed these as-grown TiO 2 nanotubes to be amorphous phase, mainly TiO 2 NxH 2 O [13]. After the Ag deposition, the surface topography of these as-grown TiO 2 nanotubes changed with the extent depending on the diameter. For the 100-nmdiameter sample (Figure 1F), the original nanotubular structure was almost completely retained except that the tube opening slightly shrank due to the decoration of the Ag nanoparticles. The influence of Ag decoration on the nanotube topography became more significant with a deceasing nanotube diameter ( Figure 1D and 1E). For the smallest diameter (25 nm) nanotubes, some openings were fully covered with Ag nanoparticles, resulting in more irregular topography on a nanometric scale. Figure 2 shows the TEM analysis results for a 100-nm-diameter Ag-decorated TiO 2 nanotube. Based on the atomic plane spacing in high-magnification TEM images ( Figure 2B and 2C) and EDS spectrum ( Figure 2D), metallic Ag nanoparticles were confirmed to be loaded into the TiO 2 nanotube. The Ag nanoparticles were mainly distributed near the nanotube surface, while some were incorporated into the inner surface of the nanotubes (Figure 2A). This scenario is reasonable since Ag nanoparticles have a particle size ranging from 5 to 20 nm, which is much smaller than the nanotube opening. The XPS spectra at different depths using a constant Ar + sputtering rate ( Figure 3) further compared the Ag distribution in the TiO 2 nanotubes of different diameters. The binding energies of the Ag 3d peak at 368.25 and 374.25 eV could be assigned to 3d 5/2 and 3d 3/2 of metallic Ag 0 , respectively [27], indicating that the Ag nanoparticles existed in the Ag 0 state in the TiO 2 nanotubes. We found that, compared to the nanotubes with larger diameters, the 25-nm-diameter Ag-decorated nanotubes showed a relatively strong Ag signal near the surface, indicating that the Ag nanoparticles had mainly aggregated near the nanotube surface and thus caused the more irregular topography seen in Figure 1D.

Results and Discussion
It has been reported that cell attachment, spread, and cytoskeletal organization are apparently greater on hydrophilic relative to hydrophobic surfaces [28]. Das et al. also indicated that a low contact angle means high surface energy, which is a crucial factor contributing to better cell attachment [29]. It was thus essential to study the surface wettability of the Ag-decorated TiO 2 nanotubes. In our previous study, all as-grown TiO 2 nanotubes with different diameters were highly hydrophilic as their contact angles were quite small ( Figure 4A-C). Nevertheless, after the Agdecoration process, these nanotube samples became poorly hydrophilic and their contact angles increased with an increasing diameter ( Figure 4D-F). This phenomenon can be explained by Wenzel's model [30], in which an increase of surface roughness in hydrophilic material will result in a smaller contact angle, and water will fill the grooves below the droplet. Hence larger diameter nanotubes, having smaller geometric roughness, are thought to exhibit poorer hydrophilicity. However, once irradiated with UVlight for 1 h, the Ag-decorated TiO 2 nanotubes recovered their hydrophilicity to an extent depending on the diameter (Figure 4G-I). TiO 2 is a photosensitive material, and when irradiated with UV light the photo-generated holes react with lattice oxygen to form surface oxygen vacancies to which water molecules kinetically coordinate, thereby greatly improving the surface hydrophilicity. In particular, the Ag-decorated TiO 2 materials in this study provided better separation between electrons and holes [31], further promoting the generation of oxygen vacancies and surface wettability. Figure 5 shows the cumulative Ag ion release profiles from the Ag-decorated nanotubes of different diameters into PBS solution. Both the 25-and 100-nm-diameter Ag-decorated nanotubes initially showed a higher Ag ion release rate, with a gradual  decrease in release with immersion time. However, there were no statistical differences among the Ag-decorated samples with regards to the nanotube diameter. The initial phase after implantation is the most dangerous and prone to infection, and a higher Ag ion release rate in the early stage is required to avoid post-operative infections and guarantee normal wound healing. Hence the Ag ion release profile and rate shown in this study met the clinical requirements.
The ability of Ag-decorated TiO 2 nanotubes to prevent viable bacteria colonization was verified by antibacterial tests as shown in Figure 6. The amount of viable bacteria was apparently smaller on the Ag-decorated samples compared to the as-grown samples ( Figure 6D-F). This result indicates that the decoration of Ag nanoparticles on TiO 2 nanotubes effectively inhibited the growth of bacterial colonies and showed enhanced antibacterial activity. However, there were no statistical differences among the Agdecorated samples with regards to the diameter. SEM examinations were performed to further investigate the influence of Ag decoration of TiO 2 nanotubes on the bacterial morphology. As shown in Figure 7, numerous bacteria tended to aggregate in groups on the as-grown nanotubes, while only a few bacteria were seen on the Ag-decorated nanotubes. In addition, the S. aureus displayed smooth and intact bacterial membranes on the Ag-free samples (inset in Figure 7C), while obvious membrane shrinkage and deformation were observed on the Ag-decorated samples (arrow in inset in Figure 7F). These observations indicate that Agdecorated TiO 2 nanotubes do indeed exhibit enhanced antibacterial activity against S. aureus.
The bactericidal mechanism of Ag is not fully understood, however it is generally recognized that Ag ions simultaneously attack multiple sites within the microorganism to inactivate several critical physiological functions such as cell wall synthesis, membrane transport, nucleic acid synthesis and translation, protein folding and function, and electron transport [32,33]. It is thought that Ag ions tend to have a higher affinity to react with negatively charged side groups of the proteins on the bacterial membrane such as phosphorus and sulfur compounds [34,35], which results in alteration of the molecular structure and destruction of the bacterial membrane [36]. Due to the destruction of the cell membrane, Ag ions are able to invade the bacteria, reacting with thiol groups of vital enzymes [37] and combining with DNA to disable the replication ability [38], and eventually causing the death of the bacteria. In addition, Ag ions can produce reactive oxygen species (ROS) which causes significant damage to cell structures [39]. The multifaceted bactericidal mechanisms of Ag may furnish Ag-decorated TiO 2 nanotubes with a broad spectrum of antibacterial activity.
The human fibroblast cell behavior in response to the Agdecorated TiO 2 nanotubes was studied, since the issue of cytotoxicity is crucial for practical implantations. To evaluate the fibroblast cell attachment on the TiO 2 nanotubes, cytoskeleton actin was stained with rhodamine phalloidin which expressed red fluorescence, and the nuclei were stained with DAPI which expressed blue fluorescence. The actin immunostaining showed very different cell-material contact morphology for the nanotube samples with different diameters (Figure 8). For both as-grown and Ag-decorated nanotubes, and especially the Ag-decorated series, there were much longer and well-defined actin fibers on the fibroblasts cultured on 25-nm-diameter nanotubes relative to the larger ones. It is known that cells have to adhere to a material surface first and then spread for further cell division. Better cell adhesion leads to more activation of intracellular signaling cascades through integrin coupled to the actin cytoskeleton [40,41], and hence the smaller diameter nanotubes, even those decorated with Ag nanoparticles, had more focal points for the fibroblast cells to get attached and thus aid in cell adhesion. FE-SEM was employed for detailed observations of cell adhesion ( Figure 9). The fibroblasts on the 25-nm-diameter nanotubes revealed good cell adhesion with an elongated flattened morphology, while those on the 50-nm-diameter or larger nanotubes show more rounded morphology and lack of cell spreading. It has also been reported that cells recognize surface features when a suitable site for adhesion has been detected. The cells then stabilize the contact by forming focal adhesions and mature actin fibers, followed by the recruitment of tubulin microtubules [40]. The actin cytoskeleton has been linked to integrins which are located within the adhesions. Our findings suggest that the cytoskeleton on the smaller diameter nanotubes, even those decorated with Ag nanoparticles, was formed more successfully than that on the larger diameter nanotubes. It should be noted that the aforementioned difference in surface wettability may also have contributed to and even enhanced the difference in cell adhesion between the Ag-decorated samples.
To further quantitatively evaluate the cell proliferation on the TiO 2 nanotubes, the WST-1 assay was used after cell seeding on the samples for 3 days. Figure 10 shows the comparison of optical densities measured from the WST-1 assay. We found that both asgrown and Ag-decorated nanotubes exhibited a monotonically Figure 11. XPS surface analysis results, in terms of spectra for N 1s of the as-grown and Ag-decorated TiO 2 nanotubes. All nanotube samples of different diameters, either as-grown or Ag decorates series, had much higher N 1 s intensity than the plain Ti foil. doi:10.1371/journal.pone.0075364.g011 increasing trend in cell proliferation with decreasing nanotube diameter, indicating that the fibroblast cells showed an obvious diameter-dependent behavior on both as-grown and Ag-decorated nanotubes. We also found that cell proliferation on the smallest diameter nanotubes was enhanced in comparison to the plain Ti foil. Further, the 25-nm-diameter Ag-decorated samples showed a higher optical density compared to their as-grown counterparts. This result not only indicates that the Ag nanoparticles do not exhibit cytotoxicity through an appropriate release mechanism, but also that by properly controlling the structural parameters of Ag-loaded TiO 2 nanotubes, both enhanced biocompatibility and antibacterial activity can be achieved. It has been reported that the predicted size of surface occupancy by the head of an integrin heterodimer composed of a b-propeller of the a-chain and the A domain of the b-chain is about 10 nm as estimated from electron micrographic images of individual integrin molecules [42]. It has been suggested that a 15-25 nm spacing facilitates clustering of integrins into nearly the closest packing possible, resulting in optimal integrin activation. We speculate that the decoration of Ag nanoparticles modified the surface topography of the 25-nmdiameter nanotubes, causing more irregular topography on a nanometric scale. Such a highly irregular topography may provide more suitable nanometric sites for integrin clustering, thus resulting in enhanced cell proliferation. On the other hand, nanotube diameters larger than 50 nm, which almost retained the original tubular size with the Ag incorporation, severely hindered cell spreading, adhesion, and completely prevented integrin clustering and the formation of focal adhesion complexes, eventually resulting in dramatically reduced cell proliferation. Recently, Zhao developed hybrid poly(N-hydroxyethylacrylamide) (polyHEAA)/salicylate (SA) hydrogels that combine the advantages of both antifouling and antimicrobial materials [43]. Such dual functional hydrogels, exhibiting high surface resistance to both cell adhesion and bacteria attachment, hold great potential for biomedical applications such as wound dressing and other selfhealing materials, while the Ag-decorated TiO 2 nanotubes fabricated in the present study that enhance biocompatibility and simultaneously boost antibacterial activity could be potential promising materials for medical implants. Figure 11 further shows the XPS surface analysis results, in terms of spectra for N 1 s, of all samples after immersion in the cell culture medium for 5 minutes. It shows that all of the nanotube samples with different diameters, either as-grown or Ag-decorated, had much higher N 1 s intensity than the plain Ti foil. In other words, a higher protein content existed on the nanotube samples relative to the plain Ti foil. However, there were no statistical differences among the as-grown and Ag-decorated nanotube samples. This also indicates that the decoration of Ag nanoparticles had no negative effects on the protein adsorption ability of the TiO 2 nanotubes. It has been shown that increased protein adsorption promotes osteoblastic adhesion via an enhanced interaction between cellular integrins and proteins [44]. This would promote all of the cell responses, including cell adhesion, cell spreading, and cell proliferation.
These results demonstrate that decorating TiO 2 nanotubes with Ag is a feasible scheme for fabricating implants that exhibit antibacterial activity and enhanced biocompatibility. In particular, the stability of the Ag-decorated TiO 2 structure in the physiological environment is excellent because of its inorganic nature. Most importantly, the fabrication process for Ag-decorated TiO 2 nanotubes is easy, low-cost, and suitable for industrial production. We also believe that the biocompatibility can be further tailored by optimizing the structural parameters or modifying the fabrication process of Ag-decorated TiO 2 nanotubes. For example, Ag nanoparticles can be incorporated into TiO 2 nanotubes by immersion in AgNO 3 solution followed by UV light radiation. Process parameters such as the AgNO 3 concentration, immersion time, and additives can be optimized to increase the loading amount of incorporated Ag. In addition, nanometric Ag nanoparticles with adjustable sizes and shapes can be synthesized [45] and then incorporated into the TiO 2 nanotubes more effectively to yield long-lasting antibacterial effects. These experiments in our laboratory are still ongoing.

Conclusions
In this study, Ag is electron-beam evaporated to modify the topography of anodic TiO 2 nanotubes of different diameters to obtain an implant with enhanced antibacterial activity and biocompatibility. We found that highly hydrophilic as-grown TiO 2 nanotubes became poorly hydrophilic with the decoration of Ag nanoparticles; however they could effectively recover their wettability to some extent after UV light irradiation. The results obtained from antibacterial tests suggested that the Ag-decorated TiO 2 nanotubes could greatly inhibit the growth of S. aureus. In vitro biocompatibility evaluation indicated that fibroblast cells exhibited an obvious diameter-dependent behavior on both asgrown and Ag-decorated TiO 2 nanotubes. Most importantly, of all samples, the smallest diameter (25 nm) Ag-decorated nanotubes exhibited the most obvious biological activity in promoting adhesion and proliferation of human fibroblasts, and this activity could be attributed to the highly irregular topography on a nanometric scale of the Ag-decorated nanotube surface. These experimental results demonstrate that by properly controlling the structural parameters of Ag-loaded TiO 2 nanotubes, an implant surface can be produced that enhances biocompatibility and simultaneously boosts antibacterial activity.