Layer-Number Dependent Antibacterial and Osteogenic Behaviors of

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Layer-Number Dependent Antibacterial and Osteogenic Behaviors of Graphene Oxide Electrophoretic Deposited on Titanium Jiajun Qiu, Hao Geng, Donghui Wang, Shi Qian, Hongqin Zhu, Yuqin Qiao, Wenhao Qian, and Xuanyong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00314 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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ACS Applied Materials & Interfaces

Layer-Number Dependent Antibacterial and Osteogenic Behaviors of Graphene Oxide Electrophoretic Deposited on Titanium Jiajun Qiu, †, § Hao Geng, †, § Donghui Wang, †, § Shi Qian, † Hongqin Zhu, † Yuqin Qiao, † Wenhao Qian,* ‡ and Xuanyong Liu*† †

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China ‡

Shanghai Xuhui District Dental Center, Shanghai 200032, China

§

University of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: titanium, cathodal electrophoretic deposition, antibacterial, graphene oxide, osteogenic

ACS Paragon Plus Environment

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ABSTRACT: Graphene oxide has attracted widespread attentions in the biomedical fields due to its excellent biocompatibility. Herein we investigated the layer-number dependent antibacterial and osteogenic behaviors of graphene oxide in biointerfaces. Graphene oxide with different layer numbers was deposited on the titanium surfaces by cathodal electrophoretic deposition with varied deposition voltages. The initial cell adhesion and spreading, cell proliferation and osteogenic differentiation were observed from all the samples using rat bone mesenchymal stem cells. Both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were used to investigate the antibacterial effect of the modified titanium surfaces. Co-cultures of human gingival fibroblasts (HGF) cells with Escherichia coli and Staphylococcus aureus were conducted to simulate the conditions of the clinical practice. The results show that the titanium surfaces with graphene oxide exhibited excellent antibacterial and osteogenic effects. Increasing the layer-number of graphene oxide resulted in the augment of reactive oxygen species levels and the wrinkling which led to the antibacterial and osteogenic effects respectively. Compared to pure titanium surface in the cells-bacteria co-culture process, the modified titanium surfaces with graphene oxide exhibited higher surface coverage percentage of cells.


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1. INTRODUCTION Graphene is a single-atom thick, two-dimensional hexagonal lattice honeycomb structure of sp2 hybridized carbon atoms isolated from its parent material, graphite.1-3. Thanks to the unique two-dimensional conjugated structure, graphene exhibits many exceptional chemical and physical properties including excellent electronic, thermal, optical, mechanical properties and large specific surface areas.4-12 It has attracted widespread attentions for its unique properties. With the growing research interests, graphene families (GFs), including few-layer graphene, graphene ribbons and dots, graphene oxide (GO), graphene nanosheets and flakes and reduced graphene oxide (rGO) are widely studied. Recently, graphene and its derivatives have received increasing attentions for their biological and medical applications. Substantial researches have been devoted to exploring the antibacterial activities13-17 and tissue engineering applications of GFs.18-20 To our knowledge, the physicochemical properties of GFs, such as shape,13, 21 size,22, 23 and surface functionality24 can affect their antibacterial activities. To this day, several possible mechanisms such as nanoknives, oxidative stress, and wrapping or trapping, have been proposed. However, the antibacterial mechanisms remain controversial for inconsistent experimental designs.25 On the other hand, graphene and its derivatives get a lot of attentions in the tissue engineering. Nayak et al. firstly reported that graphene could act as a promising biocompatible scaffold and didn’t inhibit the cell proliferation of human mesenchymal stem cells and guided the stem cell differentiation.26 Wu et al. produced GO-modified β-tricalcium phosphate (β-TCP-GRA) bioceramics and discovered that, compared to β-TCP control groups, β-TCP-GRA scaffolds caused an increasing rate of new bone formation in vivo.27 In terms of tissue engineering, graphene has been demonstrated as a


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biocompatible substrate for the promotion of growth and differentiation of various stem cells such as human mesenchymal stem cells, human neural stem cells, and mouse induced pluripotent stem cells.28 However, the mechanism behind graphene’s biological effect is still obscure and requires further study. Electrophoretic deposition (EPD) is a colloidal process and has advantages of simple apparatus, short formation time, and little confinement of the geometry of substrate. The thickness and morphology of deposited films can be regulated through simply adjusting the applied electric field. The charge on the colloids and the electrophoretic mobility of the colloids in the solvent is the main driving force for EPD under the influence of an applied deposition voltage.29 In this work, we fabricated different layer numbers of GO via EPD with varied deposition voltages on titanium. As the number of GO layers grew with varied deposition voltages, the wrinkling and the film roughness increased. Finally, the antibacterial activity and osteogenic ability were investigated systematically in vitro.

2. MATERIALS AND METHODS 2.1. Specimen preparation. Pure titanium plates with the dimensions of 10 × 10 × 1 mm underwent pickling with HF and HNO3 mixed aqueous solution to get rid of the oxide layers and acquire homogeneous surfaces, then ultrasonically cleaned in absolute ethyl alcohol and ultrapure water. The GO (purchased from Zhejiang carbon valley materials science and technology co., LTD.) colloidal dispersion was prepared by adding 0.5 ml GO aqueous solution (6 mg/ml) with 25 mg Zn (NO3)2·6H2O and 25 mg Zn (CH3COO)2·2H2O into 250 ml ethanol and ultrasonicated for 1 h. The Zn2+ was absorbed on GO making GO positively charged and


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deposited on titanium surfaces by cathode electrophoresis with constant potentials of 40 V, 80 V, and 120 V for 1 min, which were denoted as GO-40, GO-80 and GO-120 respectively. 2.2. Surface Characterization. Field-emission scanning electron microscope (FE-SEM, S-4800, HITACHI, JAPAN) and atomic force microscope (AFM Bruker Multimode 8 system) were used to observe the sample surface topography; X-ray diffraction (XRD; D8 advance, Bruker, Germany) patterns were acquired with a Cu Kα radiation (λ=1.5411 Å). The X-ray photoelectron spectroscopy (XPS; PHI 5802, Physical Electronics Inc, Eden Prairie, MN, USA) was applied to analyze the chemical compositions and chemical states of the specimens. The Raman spectra were obtained from 100 to 3000 cm−1 using a Raman microscope system (LabRAM, Horiba Jobin Yvon, France) with an Ar-ion laser (514 nm) for excitation. The CHI760c electrochemical workstation (CHI Instruction, Inc. Shanghai) was applied to investigate the dynamic potential polarization. 2.3. Zn Ions Release. The various samples were immersed in 10 ml phosphate buffer saline (PBS) (pH = 7.4, Hyclone, USA) at 37 °C for 1 day, 4 days, 7days and 14 days. At the end of every immersion period, the release amount of Zn ions was measured by inductively-coupled plasma atomic emission spectroscopy (ICP-AES). 2.4. Surface Zeta Potentials. Surface zeta potentials of specimens were determined using a Surpass electrokinetic analyzer (Anton Parr, Austria) as described in the literature.30 More specifically, the samples with the dimension of 20 mm × 10 mm × 1mm were fastened on the sample holders. 1 mM KCl was applied as the medium and HCl and NaOH were used to adjust the pH range. During the potential measurement process, the electrolyte solution was pumped to flow along the solid surface. Because of the motion of ions in the diffusion layer, the potentials were measured in accordance with the Helmholtz–Smoluchowski equation


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ζ=

dU dP

×

η

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×Κ

ε × ε0

In which, the ζ represents the zeta potential, dU/dP is the slope of streaming potential versus pressure, for ε, ε0, k and η, they denote the dielectric constant of the electrolyte, vacuum permittivity, conductivity and electrolyte viscosity, respectively. 2.5. Cytocompatibility Evaluation 2.5.1. Cell Culture. The cytocompatibility was evaluated using rat bone mesenchymal stem cells (rBMSCs). The cell culture was performed as the same method in the literature.31 In brief, The cell culture was performed at 37 °C in a 5% CO2 incubator with the α-minimum essential medium (Minimum Essential Medium alpha-Medium, Gbico, Invitrogen Inc.) containing 10% fetal bovine serum (Hyclone, USA) and 1% antimicrobial of penicillin and streptomycin (Hyclone, USA). The cell culture medium was changed every 3 days. The cells were incubated in a trypsin/EDTA (0.25% trypsin, 0.02% EDTA) (Gibco, Invitrogen) solution for 3 min at 37 °C to detach the cells, then centrifuged for 5 min at 1000 r/min and resuspended in the α-minimum essential medium for reseeding on the various sample surfaces. Before reseeding, the specimens were sterilized in 75% ethanol for 2 h and dried in the super clean bench. 2.5.2. Cell Adhesion and Spreading Assay. A cell density with 5.0×104 cells/ml of rBMSCs were seeded on specimen surfaces for 1 h, 4 h and 24 h. After each time point, the cells were washed with PBS, fixed with 4% para-formaldehyde solution, and permeabilized with 0.1% (v/v) Triton X-100. Then, staining was conducted with DAPI and FITC-Phalloidin. Confocal images were taken using a confocal laser scanning microscope (Leica TCS SP8). 2.5.3. Cell Proliferation. The cell proliferation was assessed using the alamarBlueTM assay (AbD Serotec Ltd., UK). Specifically, 1 ml cell suspensions with a cell density of 5 × 104 cells/ml were seeded on the various specimen surfaces for 1 day, 4 days, and 7 days. When time


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was up, the specimens were gently rinsed with PBS twice, then 0.5 ml medium with 10% alamarBlueTM was added and cultured for another 2 h. Finally, the fluorescence intensity of reduced alamarBlueTM in the medium was examined with an extinction wavelength of 560 nm and an emission wavelength of 590 nm. 2.5.4. Alkaline Phosphatase Activity Assay. The alkaline phosphatase (ALP) activity assay was conducted in accordance with the method used in the literature.30 In brief, the rBMSCs with the cell density of 104 cells/ml (7 days) and 5 × 103 cells/ml (14 days) were seeded on the sample surfaces. When time was up, 4% para-formaldehyde solution was used to fix the cells. ALP staining was performed using the AS-MX phosphate, fast blue RR salt and Mayer’s Hematoxylin solution. For the sake of quantitative assay, the cells were cultured with p-nitrophenyl phosphate at 37 °C for 30 min. Then, the ALP activity was obtained by measuring the OD405. Finally, the cellular ALP levels were obtained by normalizing against the total protein contents measured by a BCA protein assay kit. 2.5.5. Extracellular Matrix Mineralization. The Alizarin Red staining was applied to investigate the Extracellular Matrix (ECM) mineralization of various sample surfaces. The rBMSCs with the cell density of 104 cells/ml (7 days) and 5 × 103 cells/ml (14 days) were seeded on the sample surfaces. When time was up, the rBMSCs were rinsed with PBS, fixed with 75% ethanol, stained with 40 mM Alizarin Red and rinsed several times with ultrapure water. For quantitative assay, 10% cetylpyridinium chloride was used to dissolve the stain from the sample surfaces. Then, the optical density at 600 nm of the dissolved stain was measured. 2.5.6 Collagen Secretion. Sirius Red staining was applied to investigate the collagen secretion of various sample surfaces. The rBMSCs with the cell density of 104 cells/ml (7 days) and 5 × 103 cells/ml (14 days) were seeded on the sample surfaces. Afterwards, the cells were rinsed with


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PBS, fixed with 4% para-formaldehyde solution, stained with 0.1% Sirius Red and rinsed several times with 0.1 M acetic acid. For quantitative assay, the stain was dissolved in the mixture containing NaOH and methanol and the optical density at 492 nm was measured. 2.6. In Vitro Antibacterial Tests. Bacteria counting and SEM observation using Staphylococcus (S. aureus, ATCC 25923) and Escherichia coli (E. coli, ATCC 25922) were applied to investigate the antibacterial activity of various sample surfaces. The samples were sterilized with 75% ethanol for 2 h and dried in the super clean bench. Then, 60 µl bacterial suspensions (107 cfu/ml) were seeded on the sample surface and cultured at 37 °C for 24 h. For SEM observation, 2.5% glutaraldehyde solution was used to fix the bacteria overnight, then the samples with bacteria were dehydrated with gradient ethanol solutions and dried with hexamethyl disilazane ethanol solution series. For bacteria counting, after culturing for 24 h at 37 °C, the samples with bacterial suspensions were collected by test tubes with 4 ml of sterile 0.9% NaCl and shook to detach the bacteria from the specimen surfaces. Then, 100 µl of aforementioned solution was transferred to an agar culture medium for further cultivation for 18 h. Finally, the bacterial colonies were counted in accordance with the National Standard of China GB/T 4789.2 protocol. The agar diffusion assay was used to identify whether zinc ions release resulting in antibacterial effect. 100 µl of 107 cfu/ml of bacteria suspensions were added to the standard tryptic soy broth (TSB) or LuriaeBertani (LB) agar culture medium and the specimens were placed on the surface of culture medium. Followed by incubating for 18 h at 37 °C. The antibacterial performance was assessed by the width of inhibition zones around the specimens, which larger width value indicates better antibacterial performance.32 2.7. Intracellular ROS Assay. The reactive oxygen species (ROS) Assay Kit was used to investigate the intracellular ROS levels of the bacteria. 2’, 7’-dichloro-dihydrofluorescein


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diacetate (DCFH-DA) can be transformed into non-fluorescent dichlorofluorescein (DCFH) by deacetylate with intracellular esterases. DFCH can be oxidized by ROS in the cells, and produced 2’, 7’-dichlorofluorescein (DCF).33 After 24 h of cultivation as mentioned above, 500 µl of 10 µM DCFH-DA was introduced into the 24-well plates. After culturing for 20 min at 37 °C, 100 µl medium was introduced to a 96-well blank plate and the fluorescence intensity of DCF was examined with an extinction wavelength of 485 nm and an emission wavelength of 535 nm.34 The ROS levels were expressed as the ratio of (Ftest − Fblank)/(Fcontrol − Fblank ), where Ftest was the fluorescence intensity of GO-40, GO-80 and GO-120 samples, Fcontrol was the fluorescence intensity of pure Ti and Fblank was the fluorescence intensity of the 24-well plate without samples and cells.35 Finally, fold increase could be obtained by normalizing against the control group of pure titanium. At the same time, cell viability was investigated using the alamarBlueTM assay. After culturing for 24 h at 37 °C, 500 µl physiological saline solution with 10% (v/v) AlamarBlue was introduced to each sample and cultured for another 2 h. Afterwards, 100 µl medium was introduced to a 96-well blank plate and the fluorescence intensity (FI) was examined with an extinction wavelength of 560 nm and an emission wavelength of 590 nm. The antibacterial ratio was calculated as follows, Antibacterial ratio =

FIcontrol -FI test ×100% FIcontrol

FItest represents the fluorescence intensity of test samples; FIcontrol represents the fluorescence intensity of pure Ti;

2.8. Tissue cells-bacteria co-culture experiment. The tissue cells-bacteria co-culture experiment was conducted according to the method as described in the literatures.36-38 To be specific, 20 µl bacterial suspensions (105 cfu/ml) of the different bacterial strains (S. aureus and

E. coli) were applied to the specimen surfaces and incubated for 90 min at 37 °C. Subsequently,


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the samples were washed with PBS for three times. Then, 2 × 104 cells/ml of human gingival fibroblasts (HGF) cells which were suspended in the modified culture medium, supplemented with 2% bacterial culture medium (TSB for S. aureus and LB for E. coli), were seeded on the aforementioned sample surfaces and then incubated for 48 h in 5% CO2 at 37 °C. After 48 h, the HGF cells were fixed with 4% para-formaldehyde, stained with DAPI and TRITC-phalloidin, and imaged with a fluorescence microscope (Olympus, Japan). The results were quantitatively analyzed based on the surface coverage rate of the samples by HGF cells in the presence of contaminated bacteria.

2.9. Statistical Analysis. The Data were expressed as the mean ± standard deviation. The statistical significance of the difference was analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. Statistical analysis was assessed using the GraphPad Prism 5 software. A value of P < 0.05 indicated statistically significant difference.

3. RESULTS AND DISCUSION 3.1. Surface Characterization. Figure 1 displays the surface topography of the pure Ti and GO modified samples designated as GO-40, GO-80 and GO-120. The pure Ti has a mirco-nano rough surface with visible heaves (Figure 1a), while wrinkles overspread the GO-40, GO-80 and GO-120 surfaces, owning to the GO spread on the sample surfaces equably (Figure 1b, c, d). With the increase of deposition voltages, the film thickness becomes thicker as it is shown in Figure 2a, b and c. The thicknesses of the GO-40, GO-80 and GO-120 films are approximately 25 µm, 90µm and 136µm respectively. Herein, the thickness of single layer of GO is a constant and less than 1 nm.39 Therefore, the increase of film thickness indicates the augment of the layernumber of GO.


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The Raman spectra obtained from the sample surfaces of GO-40, GO-80 and GO-120 present similarly (Figure 2d). The feature peaks of GO, such as D band at ~ 1350 cm-1 and G band at ~ 1580 cm-1, are intensively appeared.40 However, the Raman spectrum of titanium surface is shown as a straight line. The feature peaks of ZnO are not detected in the GO modified samples. For observing the morphology of sample surfaces, the AFM was used and the results are shown in Figure 3. The surface roughness parameters, for example, the mean roughness (Ra) and the root mean square of the Z data (Rq), were analyzed using NanoScope Analysis software. As it is shown in Figure 3 (a-1, b-1 and c-1), with the increasing layer-number of GO, the surface roughness of samples increases gradually, which the Ra and Rq of GO-40 are 9.91 nm and 12.5 nm respectively, and the mean roughness (Ra = 16.5 nm) and root mean square of the Z data (Rq = 20.3 nm) of GO-80 are higher than that of GO-40, and GO-120 has the highest mean roughness (Ra = 26.5 nm) and root mean square of the Z data (Rq = 31.2 nm) among them. At the same time, the fluctuation amplitude of wrinkles becomes bigger as displayed in Figure 3 (a2, b-2 and c-2). The red colors present the sunken districts while the purple colors present the raised districts. With the augment of layer-number of GO, the areas of sunken and raised districts are all improved and the height differences between the sunken and raised districts become larger. From Figure 3 (a-3, b-3 and c-3), we can see that the maximum wrinkle heights of GO40, GO-80 and GO-120 are 31.4 nm, 61.4 nm and 84.5 nm respectively. With respect to the phase compositions of various samples, the XRD patterns (Figure 4a) suggest that all the samples exhibit typical features of α-Ti.31 It is worthy of note that no typical features of Zn-containing compounds were observed in any specimens. To determine the element types and chemical states, all the specimens were investigated by XPS technique and the results are shown in Figure 4b. C, O, and Ti can be found on the titanium surfaces, however, Ti is not


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detected on the GO modified samples while Zn is appeared, which indirectly demonstrates that GO overspread the titanium surfaces uniformly. The Zn 2p doublet (the inset of Figure 4b) at 1021.9 eV (2p3/2) and 1045 eV (2p1/2) corresponds to the bivalent zinc. Combining the results of Raman spectra, XRD patterns and XPS spectra, it comes to a conclusion that zinc element, to a great extent, exists in the form of zinc ion which is absorbed on the GO surface by electrostatic interaction via oxygen-containing functional groups. From the Table 1, we can know that, the zinc contents on GO modified samples reduce gradually with the increase of layer-number of GO. As it is known, increasing the deposition voltages can increase the deposition rates. With time goes by, the concentration of zinc ions around the samples decrease gradually during the EPD process which results in the reduction of zinc ions contents on the sample surfaces.

3.2. Corrosion Resistance. Krishnamoorthy et al.41 demonstrated the efficiency of GO nanosheets for inhibition of Cu metal corrosion and discovered that the protective efficiency of GO modified Cu substrates can approach 70%, compared to the bare Cu substrate. It may be ascribed to that GO can restrict electron transfer and effectively inhibit metal oxidation and oxygen reduction. In this work, Potentiodynamic polarization was performed in PBS to investigate the corrosion performance. As presented in Figure 5a. The corrosion potentials of GO-40, GO-80 and GO-120 shift positively compared to pure titanium plates. With the increase of layer-number of GO, corrosion currents reduce gradually, and GO-120 exhibits the best corrosion resistance.

3.3. Surface Zeta Potential. Zeta potentials were detected to analyze the charge on the specimen surfaces. Zeta potential versus pH for all the samples is shown in Figure 5b and we can know that the zeta potential descends with the ascending value of pH in the KCl solution. GO modified


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samples presented more positively zeta potentials, compared to pure Ti at the pH of 7.4, with the increasing layer-number of GO, the zeta potentials shift positively.

3.4. Zn Ions Release. The number of zinc ions released from the GO modified specimens was investigated by ICP-AES as shown in Figure 5c. With the increase of layer-number of GO, the cumulative Zn2+ concentration reveals descending trend. This is because the lower zinc contents on the sample surfaces with the increasing layer-number of GO as mentioned above.

3.5. In Vitro Antibacterial Ability. Both S. aureus and E. coli were used to assess the antibacterial ability with bacteria counting. The results are shown in Figure 6. From Figure 6a, we can know that, compared to pure titanium, there is no visible bacterial colony can be found on modified titanium with graphene oxide. The number of both S. aureus and E. coli reduced almost 100% compared to pure titanium (Figure 6b). For observing the morphology and membrane integrity of bacteria which were cultured for 24 h on various sample surfaces, SEM was utilized and the results are presented in Figure 7. A lot of E. coli adhere to the pure Ti surfaces and gather together, and the typical morphology of E. coli with rod shape is observed. Meanwhile, the flagellums of E. coli are clearly visible. On the contrary, there are less amounts of E. coli on the GO modified samples than pure Ti, and the amount of E. coli reduces with the increasing layer-number of GO. Moreover, the bacteria are completely lysed which indicates that GO modified samples have excellent antibacterial activity against E. coli in vitro, especially for this who has larger layer-number of GO. Likewise, the S. aureus cells present a smooth surface with a spherical geometry on control group of pure titanium while cells are corrugated on the GO modified samples. With the augment of layer-number of GO, S. aureus cells are distorted badly.

3.6. Antibacterial Mechanisms. Graphene has excellent antibacterial properties which can inactivate or kill microbes via interacting with phospholipids,

42, 43


proteins,

44, 45

DNA/RNA,

46,

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etc. Substantial researches have been devoted to investigate the antibacterial activities of

graphene related materials. However, the underlying antibacterial mechanisms still remain controversial. To date, several predominant mechanisms have been proposed including nanoknives, wrapping or trapping and oxidative stress mediated with charge transfer or ROS production.25 Nanoknives effect is ascribed to the sharp edges of graphene that act as cutters to laterally incise microbial membrane, causing intracellular substances leakage and subsequent cell death.13 However, for micro/nanosheets like electrophoretic deposited GO here, they are quasi-parallel to the substrate which makes an orthogonal incision impossible. Moreover, increasing the layernumber of GO can augment the thickness of GO which weakens the nanoknives effect. Therefore, the antibacterial mechanism of nanoknives effect is not suitable here. Comparatively, GO, electrophoretic deposited on the titanium surfaces, with large area and being immobilized entirely, was difficult to wrap or trap bacteria just as dispersed graphene do. In this light, the most plausible antibacterial mechanism here is the oxidative pressure. As we know, oxidative pressure derives from two aspects including ROS production and charge transfer. Electron transfer plays an important part in the respiratory chain of bacteria and produces energy for bacteria survival. Li et al.48 reported that, for a bacteria/graphene/metal or semiconductor system, a circuit can be built, which electrons could be transferred from the bacterial membrane to the graphene and subsequently to the underlying metal or semiconductor substrate. Herein, introducing oxygen-containing groups to graphene can produce a lot of defects which will affect the electron transfer on the graphene surface. Meanwhile, increasing the layer-number of GO can increase the thickness of GO, which, at the same time, improves the resistance of electron transfer. Therefore, oxidative pressure mediated with electron transfer is unlikely to produce


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here. In order to investigate whether the ROS production leads to the bacteria death, we measured ROS levels using DCFH-DA assay. Results are shown in Figure 8a. GO, electrophoretic deposited on the titanium surfaces, has a higher ROS level than control group Ti. There exists significant difference among all the samples of Ti, GO-40, GO-80 and GO-120 (p < 0.001). The ROS levels increase with the augment of layer-number of GO. Moreover, increasing the layer-number of GO, the cell viability shows a descending trend (Figure 8b). Though there was no significant difference between GO-80 and GO-120, the samples of GO-120 exhibited lower cell viability than GO-80 .Seen in this light, increasing the layer-number of GO results in higher ROS levels which lead to the cell death. As mentioned above, zinc ions were used as additives making the GO positively charged and deposited on the titanium surfaces by cathode electrophoresis. The existence of zinc ions may participates in the antibacterial process. To identify whether zinc ions release results in antibacterial effect, agar diffusion assay was performed. From Figure 8c, we can find no inhibition zone around the samples, which implies that zinc ions release doesn’t make contribution to the antibacterial activity. Jiang et al.49 reported that E. coli and P. fluorescens could resist 2 mg/l Zn2+, exhibiting no or negligible mortality. Zinc ions release results show that the highest release concentration after immersion in PBS solution for 14 days is 0.4732 ppm far below 2 mg/l. In this view, the zinc ions are not involved in the antibacterial process indeed. The interplay between bacteria and material surfaces principally involves three steps.50 Firstly, bacteria attach to the material surfaces driven by gravitational forces, Brownian motion and so on. Secondly, bacteria adhere to the material surfaces involving reversible and irreversible adhesion. At an early state, bacteria adhere to the surface mediated by Van der Waal forces in a reversible process. Then, the irreversible process mediated by specific adhesions happens which


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can recognize the protein layers on the material surfaces. The physicochemical properties of the materials, like surface roughness, surface charge and hydrophilicity, play a great role in the process.51-53 Finally, the adherent bacteria proliferate, communicate and form biofilms. Once the biofilms have formed, they are difficult to deracinate. Compared to their planktonic counterparts, for bacteria in biofilms, it could be severalfold magnitude resistant to antibacterial agents.54 Accordingly, it is a critical issue to restrain the formation of biofilms for generating antibacterial properties of implants. Thanks to the unique physicochemical properties of the sample surfaces, as mentioned above, it is difficult for bacteria to attach or adhere on the material surfaces as shown in Figure 7. There are obviously less number of bacteria on the GO-40, GO-80 and GO120 than control group Ti. Moreover, the bacteria on the modified sample surfaces almost exist in the form of separate or planktonic counterparts while the bacteria gather together and form biofilms in the control group Ti. In a word, GO modified titanium surfaces, to some extent, can prevent the bacteria to attach or adhere in the material surfaces and then restrain the formation of biofilms. The rest of the bacteria which adhere to the material surfaces fortunately can be killed by the oxidative pressure mediated with ROS production. The antibacterial effects will be more evident by increasing the layer-number of GO.

3.7. Response of rBMSCs. In order to investigate the initial cell adhesion and spreading activity, the rBMSCs, cultured for 1 h, 4 h and 24 h, were stained with FITC as shown in Figure 9a. At the first hour, the rBMSCs on the pure Ti, GO-40, GO-80 and GO-120 samples exhibit an approximately spherical morphology with not much difference. After cultured for four hours, the amounts of F-actin on all the sample surfaces are much more than those cultured for one hour. The expressions of filopodia and lamellipodia can be seen on the various sample surfaces. When it comes to 24 h, plenty of filopodia and lamellipodia can be observed on the pure Ti, GO-40,


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GO-80 and GO-120 samples, and the rBMSCs exhibit a polygonal shape. However, the expressions of F-actin on the GO-40, GO-80 and GO-120 are not as good as those on the pure Ti. With the increasing layer-number of GO, the expressions of F-actin exhibit a slightly descending trend. The results indicate that the initial cell adhesion and spreading on the GO modified samples are not as good as pure Ti. This may be ascribed to the specific surface morphology of GO modified samples. The cell proliferation were investigated with the alamarBlueTM assay and the results are presented in Figure 9b. Cell proliferation shows no statistically difference on the four groups after culturing for 1day, After 4 days, the cells cultured on Ti show higher proliferation than that on GO modified samples (p < 0.001), and among the modified samples, GO-40 exhibits the highest cell proliferation (p < 0.1), followed by the GO-80 and GO-120. After culturing for 7 days, the trend becomes more obvious. It should be pointed out that the cell proliferation increases with the extension of culturing time on all the four group of samples. The rBMSCs, which were cultured on various sample surfaces for 7 days and 14 days, were stained with ALP staining kit. Figure 10a displays the ALP-positive areas. The GO modified samples have larger ALP-positive areas, compared to pure titanium after culturing for 7 days. With the increasing layer-number of GO, the ALP-positive areas improve, and GO-120 sample has the largest ALP-positive areas. When it comes to 14 days, this tendency becomes more obvious. The results turn out to be similar with the quantitative analysis in Figure 10b which indicates that GO modified Ti could stimulate the expressions of ALP activity. Alizarin Red staining was applied to evaluate the ECM of rBMSCs as shown in Figure 11a. The ECM mineralization for 7 days on GO-40 is slightly improved compared to control group of pure titanium, while GO-80 and GO-120 show significantly ascending ECM mineralization (p
GO-80 > GO-40. Moreover, the collagen secretion of rBMSCs on GO-120 sample has significantly enhanced than pure Ti (p < 0.001). After 14 days, the trend goes the same. The results show consistently with the quantitative analysis in Figure 12b. From Figure 12a, we also discover that the smallest unit of collagen secretion positive-area on GO modified Ti is smaller than pure Ti, the phenomenon becomes more obvious with the increasing layer-number of GO. The results indicate that the cell size of rBMSCs on GO modified Ti is smaller than that on the pure Ti. This may be ascribed to the surface morphology of GO modified Ti. Materials themselves with various properties can regulate cell adhesion and spreading, differentiation, and other cell behaviors. Cell adhesion and spreading is usually the first step when cells are seeded on the material surfaces, it has profound influence to modulate the forthcoming cell responses, like cell proliferation and cell differentiation. As the augment of layer-number of GO, the wrinkling and the film roughness increase which affect the cell adhesion on the material surfaces. The initial cell adhesion on GO modified titanium is not as good as pure Ti which influences the following cell proliferation. Surface morphology of materials can direct the cell shape, and then cell shape can affect the cell response, especially for cell differentiation. Peng et al.55 deeply studied the cell-material interaction between the single


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rBMSCs cells with isotropic microislands of circular, square, triangle and star shape and demonstrated that, after cultured for 7 days, the optimal adipogenesis and osteogenesis took place in the circular and star cells. Yao et al.56 also pointed that surface patterning can affect cellmaterial interactions such as cell differentiation. In this work, GO deposited on the titanium surfaces resulting in the wrinkled and rough sample surfaces as shown in Figure 3. The ups and downs of wrinkles may produce physical stimulus on rBMSCs and promote osteogenesis. Increasing the layer-number of GO, the wrinkling and the film roughness increase which may strengthens the cell-material interactions and improves the osteogenesis effectively.

3.8. In vitro tissue cells-bacteria co-culture study. Compared to the mono-culture using either bacteria or mammalian cells on the sample surfaces, the tissue cells-bacteria co-culture can veritably present the conditions of the clinical practice.38 The adhesion and spreading of HGF cells were determined by immunocyto-stained fluorescent images as shown in Figure 13a. After co-culture for 48 h, the pure titanium surface showed negligible living HGF cells whether contaminated with S. aureus or E. coli. However, there were large amounts of HGF cells observed on the GO modified titanium surfaces. In terms of the surface coverage with HGF cells (Figure 13b), there were almost no surface coverage of HGF cells on the pure titanium, which indicated that the presence of adherent S. aureus or E. coli affected the survival of HGF cells. However, the surface coverage percentage of GO-40, GO-80 and GO-120 contaminated with S.

aureus were 56.7%, 22.6% and 19.6% respectively. For E. coli contamination, the cell surface coverage percentage of GO-40, GO-80 and G0-120 were 55.8%, 47.9% and 38.9% respectively. There was no statistically significant difference among GO-40, GO-80 and GO-120 with the contamination of E. coli. The results showed that GO modified titanium surfaces can suppress the growth of bacteria even causing the bacterial death, and support a favorable environment for


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the cell adhesion and growth. However, with the increasing layer-number of GO, the samples exhibited slight inhibition for the cell adhesion.

4. CONCLUSION In this work, GO was deposited on the titanium surfaces by cathodal electrophoretic deposition. The layer-number of GO grows with the increasing deposition voltage which results in the augment of the wrinkling and the film roughness. GO modified titanium surfaces exhibit excellent antibacterial effects which can inhibit the attachment or adhesion of bacteria, prevent the formation of biofilm and kill the bacteria by oxidative pressure mediated with ROS production. Moreover, GO modified titanium surfaces can promote osteogenesis with the stimulus of wrinkled and rough material surfaces potentially. The antibacterial and osteogenic effects will be enhanced with the increasing layer-number of GO. Compared to pure titanium in the cells-bacteria co-culture process, the modified titanium with graphene oxide exhibited higher surface coverage percentage of cells.

AUTHOR INFORMATION Corresponding Author Xuanyong Liu. E-mail: [email protected]. Tel: +86 2152412409. Fax: +86 21 52412409. Wenhao Qian. E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS


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Financial support from the National Science Foundation for Distinguished Young Scholars of China (51525207), National Natural Science Foundation of China (31570973), Shanghai Committee

of

Science

and

Technology,

China

(15441904900,

14XD1403900)

are

acknowledged.

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Table 1. The relative surface element amounts of different samples analyzed by XPS technique. Elemental content (atomic %)

Samples

C1s

O1s

Ti2p3

Zn2p

GO-40

40.62

45.57

0.00

13.81

GO-80

57.35

34.91

0.00

7.74

GO-120

60.05

33.85

0.00

6.10


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Figure 1. Surface morphology of the various samples: (a) Ti, (b) GO-40, (c) GO-80 and (d) GO-120. 160x108mm (300 x 300 DPI)



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Figure 2. Cross-section and Thickness of films GO-40 (a), GO-80 (b), GO-120 (c) and Raman spectra (d). 170x118mm (300 x 300 DPI)


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Figure 3. AFM images of three dimensional morphology (i-1), two dimensional morphology (i-2) and depth profile (i-3). (i = a, b and c represent GO-40, GO-80 and GO-120) 140x119mm (300 x 300 DPI)



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Figure 4. (a) XRD patterns of various samples; (b) XPS full spectra and the inset is Zn2p spectra of the various samples. 150x62mm (300 x 300 DPI)


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Figure 5. (a) Potentiodynamic polarization curves of various samples; (b) Zeta potential versus pH acquired from various sample surfaces; (c) Zn ion concentration in PBS solution after immersing GO-40, GO-80 and GO-120 for 1, 4, 7 and 14 days. 198x50mm (300 x 300 DPI)



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Figure 6. (a) Photographs of re-cultivated E. coli and S. aureus colonies on agar culture plates; (b) Analysis of reduction percentages of bacteria colonies. ***p

Layer-Number Dependent Antibacterial and Osteogenic Behaviors of

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