Biological Effect of Ultraviolet Photocatalysis on Nanoscale Titanium

Article pubs.acs.org/Langmuir

Biological Effect of Ultraviolet Photocatalysis on Nanoscale Titanium with a Focus on Physicochemical Mechanism Jingyi Wu,† Lei Zhou,*,† Xianglong Ding,† Yan Gao,† and Xiangning Liu‡ †

Center of Oral Implantology, Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, China The First Affiliated Hospital of Jinan University, Guangzhou, China

Downloaded by UNIV OF GEORGIA on September 1, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.langmuir.5b01850

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ABSTRACT: Physicochemical properties, regulated by various surface modifications, influence the biological performance of materials. The interaction between surface charge and biomolecules is key to understanding the mechanism of surface−tissue integration. The objective of this study was to evaluate the biological response to a nanoscale titanium surface after ultraviolet (UVC, λ = 250 ± 20 nm) irradiation and to analyze the effects via a physicochemical mechanism. The surface characteristics were evaluated by field-emission scanning electron microscopy, X-ray photoelectron spectroscopy, surface profilometry, and contact angle assay. In addition, we applied the zeta-potential, a direct method to measure the electrostatic charge on UV-treated and UV-untreated titanium nanotube surfaces. The effect of the Ti surface after UV treatment on the biological process was determined by analyzing bovine serum albumin (BSA) adsorption and osteoblast-like MG-63 early adhesion, morphology, cytoskeletal arrangement, proliferation, and focal adhesion. Compared to an anodized titanium nanotube coating, UV irradiation altered the contact angles on the control surface from 51.5° to 6.2° without changing the surface topography or roughness. Furthermore, titanium nanotubes after UV treatment showed a significant reduction in the content of acidic hydroxyl groups and held less negative charge than the anodized coating. With regard to the biological response, along with an enhanced capability to adsorb BSA, osteoblasts exhibited higher colonization and viability on the UV-treated material. The results suggest that UV treatment enhances the biocompatibility by reducing the electrostatic repulsion between biomaterials and biomolecules.

1. INTRODUCTION Titanium, with its favorable biocompatibility, has been widely used in all aspects of clinical medicine, being used, for example, as a component of biomaterials for the treatment of bony defects and for production of cardiovascular stents.1 Since it is known that surface physicochemical properties play important roles in biointegration, researchers have devoted their studies to modifying titanium substrates, targeting alterations of the surface topography and surface chemistry, in order to activate bioinert titanium.2,3 In terms of surface topography modification, conventional microscale titanium surface treatments such as sandblasted acid etching4 and microarc oxidation5 have been thoroughly studied and extensively applied. These microscale surface modifications have indicated that better osseointegration can be achieved by adjusting the surface topography of titanium. However, several studies have demonstrated that microscale surface treatments limit cell attachment and proliferation.6,7 Therefore, to meet the demand for immediate loading in current clinical practice, a requirement for modification of biological properties to enhance biointegration has been raised for biomaterials. Recent studies have suggested that nanostructured titanium may better mimic the surface features of natural bone, thus providing a more beneficial surface−cell interface for cellular activities.8 Hence, a surface coating with nanoscale structure © XXXX American Chemical Society

offers a good opportunity to make biomaterials more functional. Different nanoscale surfaces can be processed using different treatment techniques,8,9 and various nanowires,10,11 nanopits,12 nanopores,13 nanonodules,14 and nanotubes15,16 have been studied for their effects on cellular behaviors. Nanotubes, a specific nanostructure, have drawn the most attention. These structures not only greatly enhance the surface area but also possess ample tube-like space for loading functional drugs.17 Oh et al. reported that cells exhibited better differentiation efficiency on nanotubes with diameters of 70−100 nm.18 In our previous study, we found that titanium nanotubes with a tube diameter of 80−100 nm tended to be more beneficial in promoting differentiation into osteoblasts and resulted in better early osseointegration around the implant. In this study, we synthesized nanotubes using the most suitable nanotube diameter identified under previous experimental conditions. UV photocatalysis, a unique surface property of semiconductors like titanium, has been used as a method of surface modification. It can improve the biological response to titanium without changing surface features. Except for its disinfection function, UV-induced superhydrophilicity of titanium oxide was Received: May 25, 2015 Revised: July 23, 2015

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DOI: 10.1021/acs.langmuir.5b01850 Langmuir XXXX, XXX, XXX−XXX

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Filderstadt, Germany). This involved dropping 10 μL of distilled H2O onto the titanium surface and immediately photographing the magnified side view image of the droplet using SCA20 software (Dataphysics). The contact angle was determined by randomly choosing five points from each disk. 2.3. Zeta-Potential. Zeta-potential data inferred from streaming potential were measured on both UV-treated titanium disks and untreated titanium surfaces, applying a constant gap (0.1 mm) between two rectangular samples with the same surface treatment. The electrokinetic streaming potential (Anton Paar, Graz, Austria) was automatically surveyed in forward and backward flow directions. The measurements were performed in 0.001 mol/L KCl solution, and the pH was varied from 3 to 8 by addition of 0.1 mol/L HCl or 0.1 mol/L NaOH. For statistical analysis, four streaming potentials were obtained at each pH value. 2.4. Protein Adsorption Assay. Bovine serum albumin fraction V (BSA, Genview, El Monte, FL) was used as a model protein. Normally the protein solution of BSA was prepared according to a classical protocol, using PBS at pH 7.4. A 300 μL droplet of protein solution (1 mg/mL in PBS) was added over titanium disks. After 3 or 24 h of incubation in sterile humidified conditions at 37 °C, the samples were transferred to a new 24-well plate and washed thrice with PBS; then 500 μL of 1% sodium dodecyl sulfate (SDS) solution was introduced into the wells, and the plate was shaken for 1 h at constant speed on an orbital shaker (TS-100, Qilinbeier, Jiangsu, China) to detach proteins from the samples. A 100 μL aliquot of the collected solution was mixed with 100 μL of microbicinchoninic acid (Pierce Biotechnology, Inc., Rockford, IL) in a new 96-well plate and incubated at 37 °C for 1 h. The optical density (OD) of each disk was quantified using a microplate reader (680, Bio-Rad, Hercules, CA) at 595 nm. The amount of protein adsorption was calculated using a standard curve, obtained by plotting the average blank-corrected 595 nm reading for each BSA standard from the kit vs its concentration in μg/mL. 2.5. Cell Culture. Human osteoblast-like MG-63 cells, derived from a human osteosarcoma (CRL-1427, ATCC, Manassas, VA), were cultured in Dulbecco’s minimum essential medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Thermo Scientific, Pittsburgh, PA) and 1% penicillin streptomycin (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. When the cells reached 80% confluence after 4 days of incubation, they were detached using 0.25% trypsin−1 mM ethylenediaminetetraacetic acid (EDTA)−4 Na (Gibco) and seeded onto substrates at a density of 1.0 × 104/cm2 or 2.4 × 104/cm2. The culture medium was renewed every other day. 2.6. Cell Adhesion Assay. The cells were seeded onto the entire titanium disk surface in a 24-well plate at a density of 1.0 × 10−4/cm2. After incubation for 0.5, 1, and 2 h, the cell suspension was removed. The samples were transferred into a new 24-well plate and then washed thrice with PBS to remove the nonadherent cells, fixed in 4% paraformaldehyde for 30 min at room temperature, and finally stained with Hoechst (10 μg/mL, 33342, MP, Solon, OH) in the dark under ambient conditions. The number of dyed nuclei was counted under a fluorescence inverted microscope. Each sample was selected to acquire a comparative of 10 random well-distributed fields, magnified 100×. The cell numbers were counted using the software Image-Pro Plus 6.0 (IPP, Media Cybernetics, Rockville, MD). 2.7. Cell Morphology and Morphometry. Cell morphology and cytoskeletal structure were observed by confocal laser scanning microscopy (710NLO Zeiss, Jena, Germany) with a suitable objective lens such as Plan-Apochromat 20×/0.8 M27 (Zeiss) or PlanApochromat 100×/1.40 Oil DIC M27 (Zeiss). The density of cells seeded onto the titanium surface was the same as used for the cell adhesion assay. After incubation for 3 and 24 h, cells on the substrates were fixed in 4% paraformaldehyde for 30 min at room temperature and rinsed thrice with PBS. The fixed cells were permeabilized with 0.1% Triton-X100 for 5 min. After blocking with 10% normal goat serum for 30 min, the cells were stained with 50 μg/mL Phalloidin-TRITC (tetramethylrhodamine B isothiocyanate) (Sigma-Aldrich, St. Louis, MO). IPP analysis software was used to measure the area, diameter, perimeter, and IOD.

first reported in Nature in 1997.19 This unique characteristic has significantly enhanced the bioactivity of microscale titanium both in vivo and in vitro.20−25 However, the efficacy of UV photocatalysis would be strengthened by increasing the surface specific area.26 This suggests that UV photocatalysis could reinforce the biocompatibility of microscale modificatory titanium, but whether or not the enhanced efficacy of UV photofunctionalization resulting from the addition of titanium nanotubes with a larger surface area will facilitate biocompatibility remains unknown. In addition, complicated physicochemical interactions occur on the titanium surface during and after UV irradiation.19,27 Hori et al.27 have proposed that the enhancement of bioactivity of UV-photofunctionalized microscale titanium correlated with the surface electrostatic property. However, the physicochemical mechanism of UV irradiation on the titanium coating with nanostructure remains to be elucidated. We therefore hypothesize that UV irradiation alters the chemical composition on the nanoscale titanium surface and surface electrostatic force. These changes act as the major driving force for the interaction between biomolecules and material substrates. This study was conducted to examine the effect of UV pretreatment of nanostructured titanium on the in vitro performance and behavior of osteoblasts on the substrates and to explore the physicochemical mechanisms underlying the effect of UV light on nanoscale titanium. We concentrated on three questions: (1) What is the effect of UV-irradiated nanotubes on biological behaviors? (2) Does UV photofunctionalization change the chemical composition of the nanotubes? (3) If so, do those alterations influence the surface charge of nanotubes? To determine the physicochemical properties of samples, we used X-ray photoelectron spectroscopy (XPS) analysis. The surface charge was investigated by solid zeta-potential which provides a direct measurement of surface electrostatic force. In order to study the biological compatibility, initial biological activities such as adsorption of protein and adhesion, proliferation, and behavior of osteoblasts were investigated.

2. MATERIALS AND METHODS 2.1. Preparation of Titanium Samples. Commercially available, pure grade 2 titanium (KS45, Sumitomo, Japan) was cut into Ti disks, 15 mm in diameter × 1 mm in thickness. The prepared titanium disks were ground with a graded series of sandpapers to remove machining scratches and ultrasonically cleaned with acetone, absolute ethyl alcohol, and distilled water successively for 20 min each. In order to produce a nanostructured surface, the titanium samples were anodized at 20 V for 20 min in an electrolyte containing 0.5 wt % hydrofluoric (HF) acid using a direct current (dc) power supply with a platinum electrode as the cathode. All of the samples were sterilized using 25 Gy γ-rays equipment for 12 h (GM-II, Gamma Beijing, Huada Biotechnology Co. Ltd.). After treatment, titanium disks were stored in the dark under ambient sterile conditions (clean room, humidity >60%, temperature >25 °C). As described in a previous study, half of the titanium substrates were treated with UVC (λ = 250 ± 20 nm) for 48 h under ambient conditions using a 15 W bactericidal lamp (Philips, Tokyo, Japan) at an intensity of 2 mW/cm2.23 The UV-untreated samples were used as controls. 2.2. Surface Characterization. The surface topography of these samples was observed by field-emission scanning electron microscopy using an accelerating voltage of 5 kV and 32 frames/s (FE-SEM, 1024 × 768, 1530 VP, LEO, Oberkochen, Germany), and surface roughness was quantified using a surface profilometer to determine the Ra and Rz values (Wyko NT1100, Bruker, Tucson, AZ). The chemical composition of the samples was investigated by XPS (ESCALAB 250, Thermo Fisher Scientific, London, UK) under vacuum conditions (∼2 × 10−9 mbar). Hydrophilicity of the titanium surface was measured using a contact angle measuring system (OCA15, Dataphysics, B


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Figure 1. FE-SEM micrographs (top views) of nanotubular topography on titanium surface with (CD) or without (AB) UV irradiation. Scale = 200 nm. Surface roughness of a nanothin TiO2 layer with (F) or without (E) UV-treatment. Quantitative measurement of two surface roughness parameters, Ra (I) and Rz (J) for AO and AO+UV disks, was based on the surface prolilometer analysis. Photographic images of 10 μL of H2O droplet on titanium nanotube surface before (G) and after (H) UV treatment as well as statistically compared contact angle of H2O (K) are exhibited. Data are mean ± SEM (n = 3). **p < 0.01 indicating a significant difference between AO and AO+UV surface. 2.8. Vinculin Expression Analysis. During the preparation for confocal laser microscopy and cell morphology analysis, the disks were stained with mouse antivinculin monoclonal antibody (Abcam, Cambridge, MA), followed by FITC-conjugated antimouse secondary antibody (Boster, Pleasanton, CA). The expression of vinculin was calculated as the number of vinculin expressions per cell and per area using IPP image analysis software. 2.9. Cell Proliferation Assays. MG-63 cell suspensions at a density of 2.4 × 104 cells/cm2 were seeded onto the titanium substrates and incubated in a 5% CO2 and 95% air incubator at 37 °C for 1, 4, or 7 days. At the end of each incubation period, the samples were rinsed thrice with PBS. Cell proliferation was assessed using the tetrazolium compound 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS, Promega, Madison, WI). Each sample was incubated in a mixture of culture medium and MTS solution at 37 °C for 3 h. The assay was performed in a 96-well plate, and the optical density was recorded at 490 nm with background absorption correction using a microplate reader (Bio-Rad). 2.10. Statistical Analyses. Each experiment was repeated three times. All data were analyzed using IBM SPSS 19.0 software (IBM, Armonk, NY) and Graphpad Prism 5. All values are presented as mean ± standard error of the mean (SEM). Statistical comparisons of all experiments were performed using a general linear model across two independent variables: group (UV-treated and UV-untreated titanium nanotube, presented in figures as AO+UV and AO, respectively) and time. Comparisons between the two groups or two groups at different time points were performed using two-sample t tests, and the P value was adjusted by the Bonferroni method. Curve fitting was used to fit the relationships between zeta-potential and pH in the two groups separately. All P-values were two-sided, a P < 0.05 was considered to be statistically significant, and P < 0.01 was considered to be highly significant.

3. RESULTS 3.1. Surface Characterization. Following the previous manufacturing methods for nanotube assembly, TiO2 nanotubes were successfully synthesized with diameters of approximately 80−100 nm. The UV-treated titanium disks showed no obvious differences in extrinsic features under FE-SEM (Figure 1A−D). Surface roughness (Sa, Rz, Figure 1E,F) did not differ markedly between the groups (Figure 1I,J). However, magnified images of the contact angle in the two groups were significantly different, as shown in Figure 1K. Figure 1G shows that a H2O droplet remained as an arc shape without spreading on UV-untreated nanotubes. In contrast, in the UV-treated group, H2O spread extensively over the entire disks (Figure 1H), indicating that UV-irradiation enhanced the hydrophilicity of the titanium nanotubes. The contact angles on UV-treated substrates were shifted from 51.5° to 6.2°. 3.2. Surface Chemical Species of Specimens. Figure 2 shows the O 1s spectra of UV-treated and UV-untreated titanium nanotube samples. The O 1s peak was resolved into three individual photopeaks.30 The major peak at 530.00 eV corresponded to the O 1s of TiO2, and the second peak located at a binding energy of 531.40 eV was assigned to the O 1s of acidic TiOH. The peak at 532.15 eV was attributed to the O 1s of basic TiOH. To compare the content of the OH group, the relative area ratios of the 3 O 1s peaks were calculated and suggested that the percentage area of the peak at 531.4 eV corresponded to 23.6% acidic hydroxyl for the titanium nanotubes and 15.6% for UV-treated nanotubes, while the peak located at 532.4 eV assigned to the basic OH group represented 9.2% for the control C


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Figure 2. O 1s spectra peak was measured by XPS for the AO and AO+UV titanium surface (A) and the acidic hydroxyl groups were shown by arrows. Percentage area of two type of hydroxyl for AO and AO+UV (B).

group and 11.1% for the UV-treated group (Figure 2B). It is worth noting that the relative area ratio of the peak at 531.4 eV for the UV-treated nanotubes was significantly less than that for the ordinary titanium nanotubes. In contrast, the area of the peak at 532.4 was higher in the UV-treated group. 3.3. Surface Charge of Specimens. Zeta-potential measurements revealed a difference in electrokinetic interactions at the interface between the biomaterial surface and the aqueous electrolyte in this study. Figure 3A shows the zeta-potential vs pH of a standard 0.001 M KCl solution for the surface of UV-treated and untreated titanium nanotubes. The curve of zeta-potential vs pH of all the samples ranged from pH 3 to 8. This clearly shows that the surface charge varies with changes in the pH of the solution. The isoelectric point, the point where the curve crosses the X-axis in Figure 3A, indicates where the positive charge on the surface is equal to the negative charge. Isoelectric points at pH 3.6 for UV-treated and pH 4.2 for UV-untreated titanium surfaces were observed. Figure 3B reveals the zeta-potential for both UV-treated and untreated titanium nanotubes at pH 7.4.

It clearly shows that UV-treated titanium nanotubes have a lower absolute value of zeta-potential compared to UV-untreated nanotubes. 3.4. Increased Albumin Adsorptive Capacity of UVTreated Titanium Nanotubes. Figure 4 shows that for both the UV-treated and untreated nanotubes the amount of protein adsorption increases with incubation time. The amount of BSA adsorbed to UV-photofunctionalized titanium during either 3 or 24 h incubation was significantly greater than that of the untreated group (Figure 4). The amount of adsorbed protein on the UV-treated titanium nanotubes after 3 h was considerably higher than that on the UV-untreated nanotubes after 24 h. Furthermore, during the incubation time, the increase in the protein adsorption in the untreated group was higher than in the UV-treated group, indicating that the UV-treated nanotubes reach the saturation in a shorter time (Figure 4B). 3.5. Enhanced Osteoblast Attachment on the UVTreated Titanium Nanotube. The adherent nuclei of MG-63 cells on each group of nanotubes are shown in Figure 5A−F. D


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Figure 3. pH dependence of zeta-potential for titanium nanotube with and without UV irradiation in 1 mmol/L KCl (A). Comparison of zeta-potential at pH = 7.4 for AO and AO+UV (B). Data are mean ± SEM (n = 4). *p < 0.05 indicating a difference between AO and AO+UV surface.

Figure 4. Amount of protein adsorption on titanium nanotube surface before and after UV treatment at each point (A) as well as the changes of protein adsorption of two groups are presented (B), indicating the increasing rate. Data are mean ± SEM (n = 3). **p < 0.01 indicating a significant difference between AO and AO+UV surface.

clearly higher than that on the control material at every time point. Figure 5H represents the rate of cell adhesion on both titanium surfaces. This result confirmed that during the first 30 min the adhesion rate onto UV-treated samples was clearly higher than in the UV-untreated group, although the rate of adhesion reduced after 1 h of incubation.

Data from the three selected time points showed that the number of adherent cells on the surface of both the UV-treated and untreated titanium nanotubes increased with time (Figure 5G). In the first 30 min of incubation, UV treatment significantly enhanced the number of adherent cells on both substrates. In addition, the cell number on the UV-treated substrates was E


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Figure 5. Fluorescent stained nuclei of MG63 cells attached on AO and AO+UV surface at 0.5, 1, and 2 h of incubation (A−F). Ten random fields were selected from every titanium disk on each group. Scale = 100 μm. Quantitative comparison of initial cell attachment on titanium surface with or without UV treatment (G). The early adhesion rate of MG63 on AO and AO+UV surface (H). Data are mean ± SEM (n = 3). *p < 0.05 indicating a difference between AO and AO+UV surface; **p < 0.01 demonstrating a significant difference between AO and AO+UV surface.

3.6. Change in Cell Morphology on Exposure to UVTreated Titanium Nanotubes. Confocal laser scanning microscopic images after staining with rhodamine phalloidin are shown in Figure 6C. The osteoblasts were observed to be markedly larger on UV-treated titanium nanotubes than on untreated surfaces after incubation for 3 h. Osteoblasts on UV-treated titanium surfaces began to spread in a spindle shape, whereas the cells on the control surfaces retained a circular morphology. After 24 h of incubation, the cells on UV-untreated disks had become elongated while those on the UV-treated surfaces were polygonal. Even though osteoblast elongation was observed on both samples after 24 h of incubation, the osteoblasts on the UV-treated nanotubes had elongated more than the cells on the control surface. Based on the analysis of microscopic images, the parameters of cytomorphology revealed significant differences between the two groups, confirming the results of the above-mentioned qualitative observations (Figure 7A−C). 3.7. Improved Expression of the Focal Adhesion Protein Vinculin. Microscopic images of antivinculin-stained cells are shown in Figure 6D. After 3 h of incubation, vinculin was distributed around the cells on both UV-treated and UV-untreated titanium nanotube surfaces, except for areas of enlarged cells. The vinculin present at the edges of cells was easily visible in cells growing on the UV-treated titanium nanotubes.

After 24 h incubation, the cells on UV-treated nanotubes exhibited more extensive expression of vinculin around the periphery of polygonal cells compared to the UV-untreated titanium nanotubes, where the vinculin on the surface was only present at the tip of projections of the spindle cells. Densitometry, based on microscopic images showed that vinculin expression, calculated either per area or per cell, was higher on UV-treated titanium nanotubes than on control substrates, although there were no significant differences in the quantitative parameters of vinculin expression between these two groups (Figure 7D,E). 3.8. Enhanced Osteoblast Proliferation on the UVTreated Titanium Nanotubes. Osteoblast proliferation was measured by MTS assay. Figure 8 shows that the optical density (OD) on UV-treated titanium surfaces was visibly higher than on the control surfaces during the incubation period, signifying that the number of living cells on UV-treated titanium nanotubes was higher than on the untreated nanotubes. Cell proliferation after 7 days of incubation indicated a significantly higher proliferation rate of the UV group compared to the control group.

4. DISCUSSION This study confirms that the bioactivity of titanium nanotubes can be improved by UV treatment. UV photofunctionalization enhances the protein adsorption capacity of titanium nanotubes F


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Figure 6. Initial behavior of MG63 on titanium with and without UV treatment. Confocal microscopic image of osteoblast after 3 and 24 h of incubation with triple fluorescent staining of rhodamine phalloidin for F-actin (red), antivinculin stained for vinculin (green), and DAPI for nuclei (blue). Scale = 10 μm.

Figure 7. Parameters of cytomorphology evaluated performed on the images of F-actin (A−C). The densitometry of vinculin expression is used to calculate the level of vinculin expression per cell and per cell area (D, E). Ten random fields were selected from every titanium disk in each group. Data are mean ± SEM (n = 3). **p < 0.01 demonstrating a significant difference between AO and AO+UV surface.

as shown by the increased amount of protein adsorbed after 3 and 24 h of incubation. In addition, the growth of protein adsorption tended to plateau after 3 h of incubation on the UV-treated nanotube, indicating that the process of protein adsorption was speeded up by UV irradiation (Figure 4). Except for the improved protein adsorption, UV treatment even

considerably promoted the initial osteoblast adhesion and colonization, as demonstrated by the increased number of osteoblasts attached after 0.5, 1, and 2 h of incubation. Importantly, UV treatment expedited the process of interaction between materials and osteoblasts. In general, in the early period of contact between titanium and osteoblasts, cells adhere to the G


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Figure 8. Optical density of attached MG63 on AO and AO+UV, evaluated by MTS assay at 1, 4, and 7 days of incubation, reflecting the activity of cell proliferation (A). Change of optical densities at each time point indicating the level of cell proliferation (B). Data are mean ± SEM (n = 3). *p < 0.05 indicating a difference between AO and AO+UV surface; **p < 0.01 demonstrating a significant difference between AO and AO+UV surface.

coordinated with Ti, also called bridge hydroxyls, and singly coordinated hydroxyl groups, which are supposed to be the basic OH and are called terminal hydroxyls.30 The relative area ratio of basic OH in UV-treated nanotubes was higher than in the control group. Simultaneously, the significant decrease observed in acidic hydroxyl groups indicated a comparative increase in terminal OH (Figure 2B). UV irradiation causes electrons in the valence band of a semiconductor to transition to the conduction band and form an electron−hole pair, and surface oxygen vacancies were created after reaction between electron holes and oxygen at bridging sites.33 We speculated that UV irradiation may break the chemical bond of −OH groups at bridging sites, resulting in a corresponding decrease in the amount of acidic hydroxyl groups, which are negatively charged. The acidic hydroxyl groups binds to cations while the basic hydroxyl groups tend to interact with anions.30,34 This means that the titanium surface with the decreased number of acidic hydroxyl sites probably holds more positive charge. Thus, UV treatment resulted in a decrease in the amount of acidic hydroxyl groups on titanium nanotubes, indicating a decrease in negatively charged groups and thus making the surface more susceptible to undergoing reaction with the positively charged molecules. This is in accordance with the work of Healy and Ducheyne,30 who considered that acidic hydroxyl is negatively charged. The results of XPS analysis were consistent with those of zeta-potential analysis, confirming both results. Zeta-potential, a parameter which describes the charging behavior at a solid−liquid interface, is measured by the streaming potential method. The definition of zeta-potential has been explained using the model of the electrical double layer which is called the Stern model.28 The solid surface charge depends on several factors such as the chemical composition of the biomaterial surface and the pH of the environment. At a fixed pH, the larger the positive values of zeta-potential, the more positive charges will be present at the surface which would therefore attract more negatively charged proteins and vice versa.29 Zeta-potential was first used to directly measure the surface charge on UV-treated and UV-untreated titanium nanotubes. Birgit et al. demonstrated a shift of isoelectric point with decreasing pore diameter of anodic alumina oxide by zetapotential measurement.35 In our work, the surface charge of the two different modified titanium surfaces at different pH values and the isoelectric point of the two groups were found to be pH 3.6 and 4.2, respectively (Figure 3). Previously, experimental data have shown that the isoelectric point of Ti6Al4 V is close to

titanium surface with a boost speed to a moderate rate. We can clearly perceive that UV treatment exhibited a faster increase in cell adhesion between 0.5 and 1 h of incubation in the cell adhesion assay (Figure 5). As well as cell proliferation, the growth rate tended to be moderate during days 4 to 7 of incubation, implying that the cells on the UV-treated titanium nanotubes reached a plateau of proliferation faster than those on the untreated titanium nanotubes (Figure 8). Furthermore, osteoblasts began to deform on the titanium surface after a few hours of simple adhesion and showed more expansion on UVphotofunctionalized titanium during the 3 and 24 h of cell adhesion (Figure 6), signifying that UV treatment also accelerated the activities of adherent cells. All the enhanced biological processes could be interpreted as a positive effect of UV treatment on titanium nanotubes. The process of osteoblast adhesion to a biomaterial surface can be divided into two stages: nonspecific and specific adhesion.31 The major factor in nonspecific adhesion is binding due to surface physicochemical properties, which plays a vital role in the initial stage of cell attachment. There are multiple surface characteristics which act to induce the adherence of osteoblasts to biomaterial surfaces. Surface topography and roughness are vital factors which can, to a large extent, promote subsequent biological activities. As mentioned in the Introduction, nanoscale topography effectively enhances extracellular matrix synthesis of adherent cells and leads to increased osseointegration,8 and nanotubes show a more reliable osseointegration as compared to conventional implant surfaces.32 However, in this study, these factors were the same in the two groups (Figure 1A−F,I,J), so that the effects of topography and surface roughness were removed. As is well-known, the membrane of osteoblasts is negatively charged, which means it tends to adhere to surfaces with a more positive charge. This study suggests that electrostatic force, another physical factor, may also play an indispensable role in initial cell attachment. To identify the physicochemical mechanisms underlying the enhanced cell adhesion on UV-treated titanium nanotubes, XPS was carried out to reflect the change of surface chemical components and revealed that the major chemical elements on UV-treated and UV-untreated titanium nanotubes were generally similar, but their chemical state was totally different (Figure 2A). Study of the O 1s peak revealed different contents of hydroxyl groups on the two different titanium surfaces. The hydroxyl peaks could be divided into two species: the peak at 531.4 eV was attributed to acidic hydroxyls which are doubly H


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Langmuir pH 4.4.36 Zeta-potential analysis in this study revealed that UV-treated titanium nanotubes hold less negative electric charge than UV-untreated tubes under physiological conditions. The result of the decreasing negative electric charge on UV-treated titanium nanotubes partly corresponds to the findings of Hori et al.,20 whose experimental data revealed that titanium was totally positively charged after UV treatment. However, we utilized the direct measurement method, while they deduced their data from indirect ion treatment. On the surface of amphoteric oxides such as Al2O3 and TiO2, the acid−base properties of the hydroxyl groups on the oxide layer govern the surface electrostatic charge in aqueous solution.37 Although we hardly have a direct proof to explicate whether the decreased negative charge was attributable to the change of surface OH groups, we hypothesized that the alteration of the surface charge might correlate to the change in the surface hydroxyl groups. In addition, UV-induced superhydrophilicity has been applied in different fields. Some attributed the hydrophilicity to changes in the surface hydroxyls,38 explaining that the UV photogenerated holes were tapped at lattice oxygen sites, breaking the bond between titanium and oxygen at bridging sites and then forming single coordinated new OH groups in situ.33 The contact angle in our study had a shift in angle from 51.5° to 6.2° (Figure 1G,H,K), which could also be correlated with the surface charge. We supposed that the change of surface hydrophilicity of the titanium nanotube surface in our results might also be correlated with the change of surface OH groups. The second step in the initial adhesion of osteoblasts is a specific reaction. Previously adsorbed proteins from the extracellular matrix (ECM) may induce cell−material interaction. Cell adhesion relies on the integration between integrin receptors on the cell membrane and Arg-Gly-Asp (RGD) sequences of ECM proteins.39 According to this theory, the enhancement of protein adsorption on the biomaterial surface would have a positive effect on subsequent cellular activities. Hence, protein adsorption is also a key step in promoting cell− material interactions. Electrostatic force is the major driving force, inducing the initial nonspecific adsorption of plasma proteins.39,40 The protein adsorption assay agreed with our hypothesis, demonstrating that there is a difference in the amount of protein adsorption. Albumin is the major plasma protein, holding an isoelectric point of about 4.7; thus, it will have a negative charge under physiological conditions.41 This study demonstrated that protein adsorption is remarkably enhanced on UV-treated titanium nanotubes compared with UV-untreated nanotubes. Furthermore, UV treatment significantly expedited the process of protein−surface adsorption, reflecting the fact that the amount of protein adsorbed onto UV-treated titanium over 3 h is greater than the total amount adsorbed onto the UVuntreated group (Figure 4). The results of our protein adsorption assay partly agreed with the findings of Hori et al.,20 who described that a positively charged surface tends to attract more protein than a negative surface. Nevertheless, we considered that UV-treated titanium nanotubes adsorbed more proteins due to the decrease in the repulsive force between the biomaterial and protein. One unexpected observation relates to the expression of vinculin by cells on the UV-treated nanotubes. Vinculin expression was found to be higher than in the control group, although the difference was not significant (Figures 6 and 7). Vinculin, a key focal adhesion (FA) protein, binds to other FA molecules such as talin and actin, ultimately creating a complex of focal adhesions which are involved in cellular signal transduction

and are important for basic cellular activities such as cell attachment, proliferation, and differentiation. On the basis of our results, we speculated that the small difference in vinculin expression between the two groups might be due to three main suppositions: First, each titanium sample shares the same nanoscale surface features, which might increase vinculin expression more than a microscale surface, resulting in a failure to discover any further slight change which might occur as a result of the UV treatment. Second, Park et al.42 considered nanotubes of 100 nm to limit the formation of vinculin; we therefore hypothesized that the reason vinculin expression did not differ significantly between the two groups could be due to topographical limitation. Third, UV treatment may act on formation of other links involved in focal adhesion, rather than up-regulating vinculin expression directly in MG-63 cells. We hypothesized that changes in the surface charge could be one of the predominant influences in the enhancement of bioactivities on titanium nanotubes. To the best of our knowledge, we are the first to apply the zeta-potential to measurement of the electrostatic force on titanium before and after UV irradiation. However, in this study we focused on the change of surface charge and hydroxyl group individually. We plan to undertake further investigate the relationship between these two factors in future work.

5. CONCLUSION We compared the biocompatibility of UV-treated and UVuntreated titanium nanotubes. Our findings suggest that UV treatment enhances a series of biological activities, including protein adsorption, cell adhesion and cell proliferation. The results of XPS and zeta-potential analysis demonstrate that the content of acidic hydroxyls is significantly decreased on UV-treated nanoscale titanium, along with an increase in the positive charge. For this reason, we suggest that UV treatment enhances the biocompatibility by reducing the electrostatic repulsion between biomaterials and biomolecules.

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AUTHOR INFORMATION

Corresponding Author

*Tel +86 20 8423 3801, fax +86 20 8443 3177, e-mail zho668@ 263.net (L.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 81170998) and the Medical Science Research Foundation of Guangdong Province (Grant No. C2012034). The authors are especially grateful to Mr. Chongyang Duan (Southern Medical University, Guangzhou, People’s Republic of China) for his contribution to data analysis and Mr. Mingyang Li (Sun Yat-sen University, Guangzhou, People’s Republic of China) for his technical guidance on the analysis of XPS.

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REFERENCES

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Biological Effect of Ultraviolet Photocatalysis on Nanoscale Titanium

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