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Ag multilayers
Applied Surface Science 473 (2019) 334–342
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Full Length Article
A comparative antibacterial activity and cytocompatibility for different top layers of TiN, Ag or TiN-Ag on nanoscale TiN/Ag multilayers
T
Mengli Zhaoa,b,1, Huanhuan Gonga,b,1, Ming Mac, Lei Donga,b, Meidong Huanga,b, Rongxin Wanc, ⁎ Hanqing Guc, Yuanbin Kanga,b, Dejun Lia,b, a
College of Physics and Materials Science, Tianjin Normal University, Tianjin 300387, China Tianjin International Joint Research Centre of Surface Technology for Energy Storage Materials, Tianjin 300387, China c Tianjin Institute of Urological Surgery, Tianjin Medical University, Tianjin 300070, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: TiN/Ag multilayers Multi-arc ion plating Cytocompatibility Antibacterial activity
Surface modification is considered to be an important approach for improving the antibacterial activity and cytocompatibility of titanium alloys. Silver has been extensively studied for improving the antibacterial ability of implants due to its powerful antibacterial activity. Therefore, in this study, TiN/Ag multilayers were deposited on the surface of medical titanium alloys by a multi-arc ion plating system with the top TiN, Ag, and TiN and Ag composite (TiN-Ag) layers. The structure and chemical characteristics of the three groups were investigated by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) respectively. Scanning electron microscopy (SEM) was used to elucidate the cross-section structures of the multilayers. The surface topography, roughness, friction coefficients, hardness and elastic modulus values for different top layers were obtained by atomic force microscopy (AFM), wear tester and nanoindenter. The biological assays, including cytocompatibility experiments and antibacterial activity, were evaluated by MC3T3-E1 cells, bovine serum albumin (BSA) and Escherichia coli (E. coli) in vitro. These results confirm that the TiN/Ag multilayers with the exposed TiN-Ag top layer show excellent mechanical properties, antibacterial properties and ideal cytocompatibility, leading to the potential application of these multilayers as body-implanted materials.
1. Introduction
surface of a titanium alloy with the top layer of TiN, Ag or TiN-Ag using a multi-arc ion plating system. Due to the superior compactness of the multilayers to the single-layer structure, TiN/Ag multilayers can improve the long-term antibacterial effect, wear resistance and hardness after implantation in the body, which is conducive to the success of the surgery [12–17]. The Ag layer in the TiN/Ag multilayer is considered to be a repository of Ag ions, which can release Ag continuously to the surface via diffusion to provide a long-term antibacterial effect. On the other hand, the TiN layer possesses high hardness, wear resistance and cytocompatibility [18]. Therefore, in this paper, TiN/Ag multilayers were designed to be deposited on medical titanium alloys to improve their mechanical, antimicrobial and cellular properties. In this work, we designed TiN, Ag or TiN and Ag composite (TiN-Ag) layers to be the top layers of the TiN/Ag multilayers. TiN as the top layer should maintain high hardness, wear resistance and cytocompatibility. However, it provides only a weak antibacterial property [19–23]. Ag as the top layer can significantly improve the antimicrobial activity, which is due to the action of Ag as an antibacterial agent, and
Titanium alloys as a kind of emerging implant material have been extensively used in orthodontics, joint replacements fields and other similar applications, because of their superior corrosion resistance, novel mechanical properties, and ideal biocompatibility [1–3]. Although titanium alloys constitute a large proportion of the body-implanted materials, these alloys also have disadvantages, such as poor wear resistance, lower hardness and poor antibacterial activity, which may lead to the degeneration of implants, diffusion of the toxic metal ions, and infection after surgery, which may result in eventual surgery failure [4–6]. All these defects limit the development of these alloys in the biomedical implant field. Although various attempts have been made to achieve good mechanical properties [7,8] or antibacterial ability [9,10] individually, it is urgent to adopt surface modifications to improve titanium alloys’ hardness, wear resistance, antibacterial activity, and cytocompatibility simultaneously [11]. In this work, TiN/Ag nano-multi layers were deposited on the
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Corresponding author at: 393#, Binshuixi Road, Xiqing District, Tianjin 300387, China. E-mail address: [email protected] (D. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.apsusc.2018.12.159 Received 18 September 2018; Received in revised form 9 December 2018; Accepted 16 December 2018 Available online 17 December 2018 0169-4332/ © 2018 Elsevier B.V. All rights reserved.
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MC3T3-E1 cell
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Fig. 1. Schematic diagram of experiment.
2. Material and methods
it has been widely used to improve the antibacterial activity of implants against a broad spectrum of microbacillary and fungal species [24]. Ag can destroy the wall membrane of a bacterial cell and cause the death of the bacterial cell, while Ag is toxic for cells when its content exceeds a certain dose [25]. Therefore, a TiN-Ag composite layer was designed for use as the top layer. Our purpose here is to decrease the amount of Ag needed to achieve the excellent antibacterial property without compromising the original biological functions and that simultaneously, enhances the mechanical properties of total multilayer systems. Different antibacterial characteristics and cytocompatibility rendered by the different relative atomic percentages of Ag were assessed systematically in vitro. Fig. 1 shows a schematic diagram of the experiments performed in this work, indicating our idea of the design, which we hoped would provide a TiN/Ag multilayer with the top TiN-Ag composite layer that is able to adsorb the highest number of cells and proteins as well as the lowest number of bacteria, compared to only the top TiN or Ag layers on the TiN/Ag multilayers.
2.1. Sample preparation Titanium alloy plates (10 mm in diameter, 2 mm in thickness) as substrates were ultrasonically cleaned in ethanol and acetone for 15 min [10,26]. And samples were synthesized using a multi-arc ion plating system (SA-6T, China), as shown in Fig. 1. First, TiN/Ag multilayers were deposited on titanium alloy plates. Titanium alloy plates were mounted on a rotatable substrate holder. And two targets (99.99% titanium target & 99.99% silver target) were installed in the pulse DC power sources. Prior to film deposition, the chamber was pumped down to a base pressure less than 2 × 10−2 Pa. Subsequently, Ar gas was introduced into the chamber (maintain the pressure at 5.0 Pa) and the substrates were etched by Ar plasma for 15 min to clean. Throughout the deposition process the working pressure (0.5 Pa), the DC bias (=−50 V) and the pulsed bias (=−150 V, 40 Hz and duty cycle 30%) were constant. The TiN layer (LTiN = 50 nm) was prepared using titanium target in N2 (0.1 Pa) and Ar (0.5 Pa) gas for 5 min, and the Ag layer (LAg = 10 nm) was deposited in Ar (0.5 Pa) gas using an Ag target for 30 s. The TiN/Ag multilayers were formed by 335
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4.0 × 103 cells/mL was seeded onto each sample in 24-well plates. After incubation for 24 h, 48 h and 72 h, 30 μL of CCK-8 mixed with 270 μL of complete culture medium was added into each sample for incubation (37 °C, 2 h). Then, 100 μL of the intermixture was transferred to a 96-well plate. The optical density (OD) was recorded by an Enzyme-linked immune metric meter (Biotek EK800) at 450 nm. After incubation for 24 h, the samples with MC3T3-E1 cells were rinsed with PBS twice and fixed with 2.5% glutaraldehyde for 8 h. Then, all samples were dehydrated in a series of gradually increasing ethanol concentrations (30, 50, 70, 90 and 100 v/v%), freeze dried, sputter-coated with thin platinum layers, and observed by SEM. The BSA solution (2 mg/mL) was prepared using Bull Serum Albumin powders (BSA, Solarbio) dissolved in tri-distilled water. Then, 1 mL of the BSA solution was added onto the samples’ surfaces and cultured at room temperature for 6 h. Next, the supernatant (2 μL) added with the loading buffer (8 μL) was tested by a polyacrylamide gel electrophoresis (SDS-PAGE USA BIO-RAD) apparatus and then dyed through Coomassie brilliant blue and destained by destainer, followed by analysis by a protein gel image analyzer (HemaGSG-2000).
alternately depositing Ti and Ag 10 times. Then, three kinds of top layers (TiN, Ag or TiN-Ag composite film) were synthesized on TiN/Ag multilayers, respectively. The top TiN layer (sample 1: top TiN on TiN/Ag) and the top Ag layer (sample 2: top Ag on TiN/Ag) on TiN/Ag multilayers were prepared in N2 (0.1 Pa) and Ar (0.5 Pa) gas using the titanium target for 12 min and the Ag target for 30 s, respectively. The top TiN-Ag composite layers (sample 3: top TiN-Ag on TiN/Ag) on the TiN/Ag multilayers were produced by codeposition using Ag target and Ti target in N2 (0.1 Pa) and Ar (0.5 Pa) gas for 12 min. 2.2. Surface characterization Scanning electron microscopy (SEM, SU8010, Hitachi, Japan) was employed to observe the structure of the multilayers. The sample for SEM observation was deposited on the single crystal silicon (1 0 0) wafer. In order to improve the adhesion strength, Ti film was pre-deposited as buffer layer on Si (1 0 0). Then the preparation processes of TiN/Ag multilayers and top layer are exactly same as above. The chemical composition of the samples was investigated using an X-ray photoelectron spectrometer (XPS, PHI5000 Versaprobe). X-ray diffraction (XRD, D8A, Bruker, Germany) with Cu-Kα (40 kV, 20 mA, λ = 1.54056 Å, Step 0.02°/s) radiation was used to characterize the microstructure of the multilayers. The surface topography, roughness and friction coefficients of the multilayers were determined by atomic force microscopy (AFM, MultiMode 8, Bruker, Germany) and wear tests (MST-4000). The hardness and the elastic modulus were characterized using a nano indenter XP system (MTS, NC, USA) with a three-sided Berkovich diamond tip using the continuous stiffness measurement method.
2.5. Antibacterial assay The antibacterial activities of the top TiN on TiN/Ag, top Ag on TiN/ Ag and top TiN-Ag on TiN/Ag were analyzed by Gram negative bacteria Escherichia coli (E. coli). Prior to the bacteria incubation, the specimens were sterilized in an autoclave at 121 °C for 40 min [31]. 1 mL bacterial suspension with a concentration of 105 cfu/mL was placed onto each sample. After incubation at 37 °C for 24 h without light, the bacteria adhering on the surfaces of samples were ultrasonically detached in the PBS solution (10 mL) for 5 min. The diluted bacteria suspension (200 μL) was introduced to a standard agar culture plate for further incubation for 24 h. The active bacteria were counted according to the National Standard of China GB/T 4789.2 protocol and the antibacterial rates were calculated as follows: Antibacterial rate (%) = [(CFU of control - CFU of experimental groups)/CFU of control] × 100%. The bacterial suspension without the samples was the control group. Different top layers all served as experimental groups.
2.3. Release of silver ions The samples (top TiN on TiN/Ag, top Ag on TiN/Ag and top TiN-Ag on TiN/Ag) were immersed in 10 mL 0.9% NaCl for various periods of time at 37 °C. The amounts of released silver ions at 7, 14, 21 and 28 days were determined by analyzing the resulting solutions using an inductively-coupled plasma atomic emission spectroscopy (ICP-AES, Horiba Jobin Yvon Ultima, Horiba, Japan).
3. Results and discussion 2.4. Evaluation of the in vitro cytocompatibility 3.1. Characterization Newborn mouse calvaria-derived MC3T3-E1 subclone 14 pre-osteoblastic cells were seeded on the surfaces of four groups to evaluate the cytocompatibility. The cells were cultured in complete culture medium containing α-minimum essential medium (α-MEM, Gibco) supplemented with 10% calf bovine serum (CBS, HyClone) and 100 units/mL penicillin and 100 μg/mL streptomycin (HyClone). The cells were maintained at 37 °C in a humidified air incubator with 5% CO2 [27,28]. They were released from the culture flask by trypsin (HyClone) and then implanted onto the samples' surfaces. MC3T3-E1 cells were seeded on the surfaces of all samples, while the inoculums density is 2 × 104 cells/mL. After 24 h culture, the cells were washed twice with the phosphate buffer saline (PBS, pH 7.4, HyClone, USA) preheated at 37 °C, and then fixed with 4% paraformaldehyde (PFA) solution (Sigma, USA) for 10 min at room temperature; the cells were then washed thrice with the PBS for 10 min every time at room temperature. The cells were also permeabilized with 0.5% (v/v) Triton X-100 (Amresco, USA) for 5 min and were then rinsed twice with PBS. Then, the cells were stained with TRITC Phalloidin (Yeasen) at room temperature in darkness for 30 min and further stained with Hoechst 33342 (Gene Copoeia) for 20 min [29,30]. Then, the F-actin and cell nuclei were examined using a confocal laser scanning microscope (OLYMPUS). Cell Counting Kit-8 (CCK-8, Genview) was used to evaluate the cells' viability and proliferation. A 1-mL cell suspension with the density of
Fig. 2A shows the SEM images (secondary electronic signal and backscattered electron signal) of the typical structure of a TiN/Ag multilayer. A modulation period (LTiN = 50 nm, LAg = 10 nm) and ten modulation periods of TiN/Ag multilayer are immediately apparent. According to the image of backscattered electron (BSE), the brighter the phase contrast represents the bigger the atomic number. Therefore, the bright layer is Ag film, and the dark layer is TiN film. In addition, the deposition rates of the Ag layer and TiN layer were approximately 20 and 10 nm/min under the constant parameters mentioned above respectively, which is also consistent with analyses by SEM. Besides, we found that the interface of Ag and TiN layers is not very sharp, most likely because no shutter is provided to periodically shelter from Ag or TiN targets during multilayer deposition. On the other hand, diffusion phenomena can easily occur at the interfaces due to the higher energy of Ag+ and Ti+ during the deposition using a multi-arc ion plating technique. The XPS depth profiles also confirm that the mixed zone is existed between TiN and Ag, as shown in Fig. 2B, because the Ag not merely exists in Ag film but exists in TiN film in the TiN/Ag multilayer. In Fig. 2B, the XPS depth profiles also show the Ag atoms on the top TiN-Ag layer are approximately 3%, and the evident diffusion layers occur when the thickness of TiN-Ag film reaches 100 nm (Ag concentrations increase along with the diffusion amount of silver diffusion). From the periodic change of Ag, the multilayer structure of TiN/ 336
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Fig. 2. (A) SEM images (SE and BSE) of TiN/Ag nano-multilayer; (B) XPS depth profiles of Top TiN-Ag on TiN/Ag; (C) the XPS survey spectra; (D) high resolution Ag3d XPS spectra of different top layers. Table 1 The relative atomic percentage of Ag for different top layers. Groups
Samples
Ag atm.%
Test group 1 Test group 2 Test group 3
Top TiN on TiN/Ag Top Ag on TiN/Ag Top TiN-Ag on TiN/Ag
0.31 27.6 2.72
Notes: Measured by XPS. Abbreviations: Top TiN on TiN/Ag: the top TiN layer on TiN/Ag multilayer; Top Ag on TiN/Ag: the top Ag layer on TiN/Ag multilayer; Top TiN-Ag on TiN/Ag: the top TiN-Ag layer on TiN/Ag multilayer.
Ag multilayer is observed, and the modulation period is about 60 nm which is consistent with analyses by SEM. Meanwhile, the elemental composition and chemical states of different samples’ surfaces were analyzed by XPS, as shown in Fig. 2C and D. Ti, N and Ag are detected for all top layers as shown in Fig. 2C, which means the mixed zones are found on the surfaces of all the samples. The high-resolution Ag3d spectrum in Fig. 2D indicates that the Ag3d3/2 and Ag3d5/2 peaks are located at approximately 373.8 eV and 367.9 eV, respectively [32,33]. The two peaks of the Ag3d doublet correspond to metallic silver. Therefore, the Ag existed in the form of metallic silver in top Ag on TiN/ Ag and top TiN-Ag on TiN/Ag. And Table 1 gives the relative atomic percentage of Ag. According to the data presented in Fig. 2C–D and Table 1, the intensity of the two Ag3d doublet peaks increases with increasing atomic fraction of Ag. Fig. 3 shows the XRD patterns of the multilayers with three different top layers. All samples display strong a preferred orientation of TiN
Fig. 3. XRD patterns of different top layers.
(1 1 1), a weaker TiN(2 0 0) and TiN(2 2 0). Additionally, they also exhibit the signature of polycrystalline Ag(1 1 1). All the samples show the crystallographic orientations of TiN and Ag, because the top layers are so thin (∼120 nm for top TiN-Ag on TiN/Ag and top TiN on TiN/Ag, ∼10 nm for top Ag on TiN/Ag) that XRD provide the information of TiN/Ag multilayers. And the mixed zones are existed in the top layers confirmed by XPS. Fig. 4A–C show the load-unload cycles of the hardness tests on three samples. The indenter displacement in each figure is more than 200 nm, which means the hardness is attributed to top layer and the TiN/Ag
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Fig. 4. The load-unload cycles of (A) Top TiN layer on TiN/Ag; (B) top Ag layer on TiN/Ag; (C) top TiN-Ag layer on TiN/Ag; (D) the hardness, elastic modulus of different top layers.
presents the highest roughness values of Ra = 8.59 nm, Rq = 13.1 nm, Rz = 6.43 nm and Rt = 123 nm. While the roughness of the top TiN-Ag layers shows the lowest values of Ra = 0.687 nm Rq = 1.22 nm, Rz = 1.31 nm and Rt = 33.7 nm. This is probably because the Ag content of TiN-Ag composite layers can reduce the average crystallite dimension of TiN. The smaller grain size gives rise to a positive effect on the mechanical, frictional, and wear properties. So, in the friction coefficients of different top layers as shown in Fig. 6A, the top TiN-Ag layer exhibits the lowest the friction coefficient of 0.143, which is due to its smooth surface. Therefore, an appropriate Ag concentration on the surface can act as a lubricant which may effectively reduce the roughness and the friction coefficient, thereby improving the wear resistance of the surface. Fig. 6B shows the amount of Ag ions released from the three kinds of samples measured by ICP-AES. There are almost no Ag ions leached from top TiN on TiN/Ag, which may be due to the Ag+ concentration below the limit of detection. And, with time, the contents of Ag ions released from top Ag on TiN/Ag and top TiN-Ag on TiN/Ag all decrease slightly. In addition, the cumulative Ag+ concentration leached from top TiN-Ag on TiN/Ag is always lower than that from top Ag on TiN/Ag as immersion time is extended from 7 to 28 days.
multilayers, and the differences reflect different aspects of the top layer. For the TiN top layer (∼120 nm), the maximum load is 12.92 mN, and the final displacement after complete unloading is about 75 nm, which means it is a hard-ceramic brittle coating. Ag is a soft metal, but the top layer is so thin (∼10 nm) that the hardness mainly reflected the information of the TiN/Ag multilayers. Therefore, the final displacement after complete unloading does not show significant increasing, although the hardness decreases. For the TiN-Ag top layer (∼120 nm), the hardness increases due to the composite structure, which is favor to inhibit the dislocation motion. The final displacement after complete unloading is close to the Ag top layer due to the toughness increasing with the Ag inclusions. Fig. 4D presents the hardness and elastic modulus values of all samples. The hardness and elastic modulus of the top Ag layer on TiN/Ag shows the lowest values (16.02 GPa and 216.2 GPa, respectively), because Ag is a soft material with a low natural hardness [34,35]. The hardness and elastic modulus of the top TiNAg layer on TiN/Ag reaches the maximum values of 30.29 GPa and 341.9 GPa, respectively. The main reason for this is that the TiN-Ag composite with the appropriate Ag content can prevent the growth of TiN columnar crystals and large particles and inhibits dislocation motion, thus generating fewer defects and resulting in improved compactness. The hardness enhancement may be attributed to suppression of dislocation movement in small grains and the narrow space between the nanocrystalline particles and amorphous phases [36,37]. The strong interfaces between nanocrystalline and amorphous phases can increase the cohesive energy of the interface boundaries, restraining the grain boundary sliding. The higher hardness can resist material deformation and damage, which is very important for the implant material. Fig. 5A–E show the 3D image and roughness, including the arithmetic mean of surface roughness (Ra), root mean square of surface roughness (Rq), ten-point height of irregularities (Rz) and maximum peak to valley height (Rt) of different top layers. The top TiN layers
3.2. In vitro cytocompatibility studies The cell initial adhesion and spreading activity on surface of samples are assayed by staining with TRITC Phalloidin (Yeasen) and Hoechst 33342 (Gene Copoeia) to visualize the F-actin cytoskeleton (red) and nuclear DNA (blue), respectively. In this paper, the MC3T3-E1 cells layers cultured for 24 h on the surface of different top were stained, and the results are shown in Fig. 7. The expressions of F-actin of MC3T3-E1 cells on the surfaces of the top TiN-Ag layer exhibits multipolar spindle morphologies with numerous filopodia and 338
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Fig. 5. 3D images of surface topography, roughness and friction coefficient of different top layers: (A) 3D images of surface topography; (B) the Ra roughness; (C) the Rq roughness; (D) the Rz roughness; (E) the Rt roughness.
After 24 h, the attached cells are observed by SEM, as shown in Fig. 9. The cells adhere tightly to the surfaces of all samples. The cells on the top TiN-Ag layer spread extensively and exhibit multipolar spindle morphologies with numerous filopodia and lamellipodia, which is consistent with the results obtained by the fluorescent assay. In addition, the cell concentrations on the top TiN-Ag layer (the low magnification SEM images) are much higher than those on the top TiN or top Ag layers, indicating that the TiN-Ag composite layer with the appropriate silver content does indeed promote the cell adhesion. When a biomaterial implanted into body, the implants initially contact with body fluids. As the most abundant protein in body fluids, BSA plays an important role in the process of cell adhesion and growth [34,35] Fig. 8B clearly shows that the top TiN-Ag layer displays the highest adsorption quantity compared to the top TiN or top Ag layers. The results for the proteins adsorption are consistent with the image of protein electrophoresis, which reflects the quantity of the remaining BSA in the protein solution after the protein electrophoresis. Therefore,
lamellipodia. The F-actin of MC3T3-E1 cells on the surface of the top Ag layer reveals a wizened structure and a small number of filopodia and lamellipodia. These results indicate that while Ag can enhance the initial adhesion and spreading activity of the MC3T3-E1 cells, excessive amounts of silver are toxic for the cells. The appropriate silver content is more favorable to the growth of MC3T3-E1 cells with no cytotoxicity, corresponding to the cell proliferation assay. The proliferation of the MC3T3-E1 cells on all top layers was assessed with cell counting Kit-8 (CCK-8, Genview) after incubation for 24 h, 48 h and 72 h. The histogram of proliferation is shown in Fig. 8A. It is clearly seen that the cell number for the top TiN-Ag layer increases gradually from 24 h to 72 h, showing the highest cell proliferation. However, by contrast, the OD values of the top TiN and top Ag layers gradually decrease. This result shows that TiN-Ag as a top layer promotes the cell proliferation. Therefore, the TiN-Ag composite layer with the appropriate silver content accelerates the cell proliferation and growth.
Fig. 6. (A) The friction coefficient; (B) Ag concentrations in 0.9% NaCl solution after immersion for 7, 14, 21, and 28 days. 339
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Top Ag on TiN/Ag
Top TiN-Ag on TiN/Ag
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Fig. 7. Confocal laser scanning microscope images of MC3T3-E1 cells adhered on different top layers: (A–C) Top TiN layer; (D–F) top Ag layer; (G–I) top TiN-Ag layer.
Fig. 8. (A) The OD values of cells on different top layers versus incubation time; (B) the protein adsorption of different top layers.
TiN-Ag layers are ∼82%, ∼89.9%, ∼99.88%, respectively. The antibacterial ability of the concerned groups is ranked as follows: Top TiNAg on TiN/Ag > Top Ag on TiN/Ag > Top TiN on TiN/Ag. These results indicate that the antibacterial property of the samples mainly depends on the surface’s metallic silver, combined with the results of released silver ions test. The smaller reduction value of E. coli for the top Ag layer than that for the top TiN-Ag layer is attributed to the content of Ag exceeding a certain range, which reduces its antibacterial property. Marques’s experiment also obtained similar results [39]. The trace silver distributed in the antibacterial materials can activate the oxygen in the air or water and produce OH− and O2−. These active groups can react with bacteria and a variety of organic compounds, which can cause the fracture of the diacetyl bond between the bases of
the top TiN-Ag layers demonstrated to show better cytocompatibility than the other top layers. The cell adhesion, spreading activity and proliferation experiments all reach the same conclusion.
3.3. Antibacterial assay To evaluate the antibacterial activities of different samples against Escherichia coli (E. coli) in the first 24 h, the spread plate method was used. The adhered bacteria were detached from the samples and recultured on agar plates in accordance with the bacteria counting methods [38]. The results are shown in Fig. 10. The amount of viable E. coli on the top TiN-Ag layers the lowest among the three different top layers. The reduction values of E. coli on the top TiN, top Ag, and top 340
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Top TiN on TiN/Ag
Top Ag on TiN/Ag
100 m
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100 m
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pseudopod pseudopod MC3T3-E1 cell pseudopod
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Fig. 9. SEM images of MC3T3-E1 cells fixed on different top layers.
Fig. 10. (A) Re-cultivated E. coli colonies on agar plates of different top layers; (B) antibacterial rates of different top layers after being incubated for 24 h.
DNA chain. Thus, the process of metabolism is disturbed and the bacteria is killed. So the Ag content is a key parameter that affects the biocompatibility and antibacterial activity of the samples. Therefore, in this paper the top TiN-Ag layer on TiN/Ag not only promotes cell proliferation but also inhibits bacteria adsorption.
grain size should be a main reason for the best biocompatibility and antibacterial activity of the top TiN-Ag composite layer. At the same time, the Ag content is a key parameter that affects the biocompatibility and antibacterial activity of the samples. Therefore, an appropriate Ag content not only promotes cell proliferation but also inhibits bacteria adsorption.
4. Conclusion
Acknowledgements
In this paper, the top TiN, top Ag, and top TiN-Ag layers on TiN/Ag are deposited on medical titanium alloys using a multi-arc ion plating system. The top TiN or top Ag layers on TiN/Ag show a certain antibacterial property but inhibit cell proliferation and growth. However, the top TiN-Ag layer on TiN/Ag shows the lowest roughness, lowest friction coefficients, lowest contact angle, and the highest hardness and elastic modulus values, leading to the best cell adhesion, spreading activity and proliferation, as well as antibacterial activities. The smaller
This work was supported by National Natural Science Foundation of China (51772209) and Program for Innovative Research in University of Tianjin (Grant No. TD13-5077). References [1] H.J. Rack, J.I. Qazi, Titanium alloys for biomedical applications, Mater. Sci. Eng. C
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Full Length Article
A comparative antibacterial activity and cytocompatibility for different top layers of TiN, Ag or TiN-Ag on nanoscale TiN/Ag multilayers
T
Mengli Zhaoa,b,1, Huanhuan Gonga,b,1, Ming Mac, Lei Donga,b, Meidong Huanga,b, Rongxin Wanc, ⁎ Hanqing Guc, Yuanbin Kanga,b, Dejun Lia,b, a
College of Physics and Materials Science, Tianjin Normal University, Tianjin 300387, China Tianjin International Joint Research Centre of Surface Technology for Energy Storage Materials, Tianjin 300387, China c Tianjin Institute of Urological Surgery, Tianjin Medical University, Tianjin 300070, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: TiN/Ag multilayers Multi-arc ion plating Cytocompatibility Antibacterial activity
Surface modification is considered to be an important approach for improving the antibacterial activity and cytocompatibility of titanium alloys. Silver has been extensively studied for improving the antibacterial ability of implants due to its powerful antibacterial activity. Therefore, in this study, TiN/Ag multilayers were deposited on the surface of medical titanium alloys by a multi-arc ion plating system with the top TiN, Ag, and TiN and Ag composite (TiN-Ag) layers. The structure and chemical characteristics of the three groups were investigated by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) respectively. Scanning electron microscopy (SEM) was used to elucidate the cross-section structures of the multilayers. The surface topography, roughness, friction coefficients, hardness and elastic modulus values for different top layers were obtained by atomic force microscopy (AFM), wear tester and nanoindenter. The biological assays, including cytocompatibility experiments and antibacterial activity, were evaluated by MC3T3-E1 cells, bovine serum albumin (BSA) and Escherichia coli (E. coli) in vitro. These results confirm that the TiN/Ag multilayers with the exposed TiN-Ag top layer show excellent mechanical properties, antibacterial properties and ideal cytocompatibility, leading to the potential application of these multilayers as body-implanted materials.
1. Introduction
surface of a titanium alloy with the top layer of TiN, Ag or TiN-Ag using a multi-arc ion plating system. Due to the superior compactness of the multilayers to the single-layer structure, TiN/Ag multilayers can improve the long-term antibacterial effect, wear resistance and hardness after implantation in the body, which is conducive to the success of the surgery [12–17]. The Ag layer in the TiN/Ag multilayer is considered to be a repository of Ag ions, which can release Ag continuously to the surface via diffusion to provide a long-term antibacterial effect. On the other hand, the TiN layer possesses high hardness, wear resistance and cytocompatibility [18]. Therefore, in this paper, TiN/Ag multilayers were designed to be deposited on medical titanium alloys to improve their mechanical, antimicrobial and cellular properties. In this work, we designed TiN, Ag or TiN and Ag composite (TiN-Ag) layers to be the top layers of the TiN/Ag multilayers. TiN as the top layer should maintain high hardness, wear resistance and cytocompatibility. However, it provides only a weak antibacterial property [19–23]. Ag as the top layer can significantly improve the antimicrobial activity, which is due to the action of Ag as an antibacterial agent, and
Titanium alloys as a kind of emerging implant material have been extensively used in orthodontics, joint replacements fields and other similar applications, because of their superior corrosion resistance, novel mechanical properties, and ideal biocompatibility [1–3]. Although titanium alloys constitute a large proportion of the body-implanted materials, these alloys also have disadvantages, such as poor wear resistance, lower hardness and poor antibacterial activity, which may lead to the degeneration of implants, diffusion of the toxic metal ions, and infection after surgery, which may result in eventual surgery failure [4–6]. All these defects limit the development of these alloys in the biomedical implant field. Although various attempts have been made to achieve good mechanical properties [7,8] or antibacterial ability [9,10] individually, it is urgent to adopt surface modifications to improve titanium alloys’ hardness, wear resistance, antibacterial activity, and cytocompatibility simultaneously [11]. In this work, TiN/Ag nano-multi layers were deposited on the
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Corresponding author at: 393#, Binshuixi Road, Xiqing District, Tianjin 300387, China. E-mail address: [email protected] (D. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.apsusc.2018.12.159 Received 18 September 2018; Received in revised form 9 December 2018; Accepted 16 December 2018 Available online 17 December 2018 0169-4332/ © 2018 Elsevier B.V. All rights reserved.
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MC3T3-E1 cell
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Fig. 1. Schematic diagram of experiment.
2. Material and methods
it has been widely used to improve the antibacterial activity of implants against a broad spectrum of microbacillary and fungal species [24]. Ag can destroy the wall membrane of a bacterial cell and cause the death of the bacterial cell, while Ag is toxic for cells when its content exceeds a certain dose [25]. Therefore, a TiN-Ag composite layer was designed for use as the top layer. Our purpose here is to decrease the amount of Ag needed to achieve the excellent antibacterial property without compromising the original biological functions and that simultaneously, enhances the mechanical properties of total multilayer systems. Different antibacterial characteristics and cytocompatibility rendered by the different relative atomic percentages of Ag were assessed systematically in vitro. Fig. 1 shows a schematic diagram of the experiments performed in this work, indicating our idea of the design, which we hoped would provide a TiN/Ag multilayer with the top TiN-Ag composite layer that is able to adsorb the highest number of cells and proteins as well as the lowest number of bacteria, compared to only the top TiN or Ag layers on the TiN/Ag multilayers.
2.1. Sample preparation Titanium alloy plates (10 mm in diameter, 2 mm in thickness) as substrates were ultrasonically cleaned in ethanol and acetone for 15 min [10,26]. And samples were synthesized using a multi-arc ion plating system (SA-6T, China), as shown in Fig. 1. First, TiN/Ag multilayers were deposited on titanium alloy plates. Titanium alloy plates were mounted on a rotatable substrate holder. And two targets (99.99% titanium target & 99.99% silver target) were installed in the pulse DC power sources. Prior to film deposition, the chamber was pumped down to a base pressure less than 2 × 10−2 Pa. Subsequently, Ar gas was introduced into the chamber (maintain the pressure at 5.0 Pa) and the substrates were etched by Ar plasma for 15 min to clean. Throughout the deposition process the working pressure (0.5 Pa), the DC bias (=−50 V) and the pulsed bias (=−150 V, 40 Hz and duty cycle 30%) were constant. The TiN layer (LTiN = 50 nm) was prepared using titanium target in N2 (0.1 Pa) and Ar (0.5 Pa) gas for 5 min, and the Ag layer (LAg = 10 nm) was deposited in Ar (0.5 Pa) gas using an Ag target for 30 s. The TiN/Ag multilayers were formed by 335
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4.0 × 103 cells/mL was seeded onto each sample in 24-well plates. After incubation for 24 h, 48 h and 72 h, 30 μL of CCK-8 mixed with 270 μL of complete culture medium was added into each sample for incubation (37 °C, 2 h). Then, 100 μL of the intermixture was transferred to a 96-well plate. The optical density (OD) was recorded by an Enzyme-linked immune metric meter (Biotek EK800) at 450 nm. After incubation for 24 h, the samples with MC3T3-E1 cells were rinsed with PBS twice and fixed with 2.5% glutaraldehyde for 8 h. Then, all samples were dehydrated in a series of gradually increasing ethanol concentrations (30, 50, 70, 90 and 100 v/v%), freeze dried, sputter-coated with thin platinum layers, and observed by SEM. The BSA solution (2 mg/mL) was prepared using Bull Serum Albumin powders (BSA, Solarbio) dissolved in tri-distilled water. Then, 1 mL of the BSA solution was added onto the samples’ surfaces and cultured at room temperature for 6 h. Next, the supernatant (2 μL) added with the loading buffer (8 μL) was tested by a polyacrylamide gel electrophoresis (SDS-PAGE USA BIO-RAD) apparatus and then dyed through Coomassie brilliant blue and destained by destainer, followed by analysis by a protein gel image analyzer (HemaGSG-2000).
alternately depositing Ti and Ag 10 times. Then, three kinds of top layers (TiN, Ag or TiN-Ag composite film) were synthesized on TiN/Ag multilayers, respectively. The top TiN layer (sample 1: top TiN on TiN/Ag) and the top Ag layer (sample 2: top Ag on TiN/Ag) on TiN/Ag multilayers were prepared in N2 (0.1 Pa) and Ar (0.5 Pa) gas using the titanium target for 12 min and the Ag target for 30 s, respectively. The top TiN-Ag composite layers (sample 3: top TiN-Ag on TiN/Ag) on the TiN/Ag multilayers were produced by codeposition using Ag target and Ti target in N2 (0.1 Pa) and Ar (0.5 Pa) gas for 12 min. 2.2. Surface characterization Scanning electron microscopy (SEM, SU8010, Hitachi, Japan) was employed to observe the structure of the multilayers. The sample for SEM observation was deposited on the single crystal silicon (1 0 0) wafer. In order to improve the adhesion strength, Ti film was pre-deposited as buffer layer on Si (1 0 0). Then the preparation processes of TiN/Ag multilayers and top layer are exactly same as above. The chemical composition of the samples was investigated using an X-ray photoelectron spectrometer (XPS, PHI5000 Versaprobe). X-ray diffraction (XRD, D8A, Bruker, Germany) with Cu-Kα (40 kV, 20 mA, λ = 1.54056 Å, Step 0.02°/s) radiation was used to characterize the microstructure of the multilayers. The surface topography, roughness and friction coefficients of the multilayers were determined by atomic force microscopy (AFM, MultiMode 8, Bruker, Germany) and wear tests (MST-4000). The hardness and the elastic modulus were characterized using a nano indenter XP system (MTS, NC, USA) with a three-sided Berkovich diamond tip using the continuous stiffness measurement method.
2.5. Antibacterial assay The antibacterial activities of the top TiN on TiN/Ag, top Ag on TiN/ Ag and top TiN-Ag on TiN/Ag were analyzed by Gram negative bacteria Escherichia coli (E. coli). Prior to the bacteria incubation, the specimens were sterilized in an autoclave at 121 °C for 40 min [31]. 1 mL bacterial suspension with a concentration of 105 cfu/mL was placed onto each sample. After incubation at 37 °C for 24 h without light, the bacteria adhering on the surfaces of samples were ultrasonically detached in the PBS solution (10 mL) for 5 min. The diluted bacteria suspension (200 μL) was introduced to a standard agar culture plate for further incubation for 24 h. The active bacteria were counted according to the National Standard of China GB/T 4789.2 protocol and the antibacterial rates were calculated as follows: Antibacterial rate (%) = [(CFU of control - CFU of experimental groups)/CFU of control] × 100%. The bacterial suspension without the samples was the control group. Different top layers all served as experimental groups.
2.3. Release of silver ions The samples (top TiN on TiN/Ag, top Ag on TiN/Ag and top TiN-Ag on TiN/Ag) were immersed in 10 mL 0.9% NaCl for various periods of time at 37 °C. The amounts of released silver ions at 7, 14, 21 and 28 days were determined by analyzing the resulting solutions using an inductively-coupled plasma atomic emission spectroscopy (ICP-AES, Horiba Jobin Yvon Ultima, Horiba, Japan).
3. Results and discussion 2.4. Evaluation of the in vitro cytocompatibility 3.1. Characterization Newborn mouse calvaria-derived MC3T3-E1 subclone 14 pre-osteoblastic cells were seeded on the surfaces of four groups to evaluate the cytocompatibility. The cells were cultured in complete culture medium containing α-minimum essential medium (α-MEM, Gibco) supplemented with 10% calf bovine serum (CBS, HyClone) and 100 units/mL penicillin and 100 μg/mL streptomycin (HyClone). The cells were maintained at 37 °C in a humidified air incubator with 5% CO2 [27,28]. They were released from the culture flask by trypsin (HyClone) and then implanted onto the samples' surfaces. MC3T3-E1 cells were seeded on the surfaces of all samples, while the inoculums density is 2 × 104 cells/mL. After 24 h culture, the cells were washed twice with the phosphate buffer saline (PBS, pH 7.4, HyClone, USA) preheated at 37 °C, and then fixed with 4% paraformaldehyde (PFA) solution (Sigma, USA) for 10 min at room temperature; the cells were then washed thrice with the PBS for 10 min every time at room temperature. The cells were also permeabilized with 0.5% (v/v) Triton X-100 (Amresco, USA) for 5 min and were then rinsed twice with PBS. Then, the cells were stained with TRITC Phalloidin (Yeasen) at room temperature in darkness for 30 min and further stained with Hoechst 33342 (Gene Copoeia) for 20 min [29,30]. Then, the F-actin and cell nuclei were examined using a confocal laser scanning microscope (OLYMPUS). Cell Counting Kit-8 (CCK-8, Genview) was used to evaluate the cells' viability and proliferation. A 1-mL cell suspension with the density of
Fig. 2A shows the SEM images (secondary electronic signal and backscattered electron signal) of the typical structure of a TiN/Ag multilayer. A modulation period (LTiN = 50 nm, LAg = 10 nm) and ten modulation periods of TiN/Ag multilayer are immediately apparent. According to the image of backscattered electron (BSE), the brighter the phase contrast represents the bigger the atomic number. Therefore, the bright layer is Ag film, and the dark layer is TiN film. In addition, the deposition rates of the Ag layer and TiN layer were approximately 20 and 10 nm/min under the constant parameters mentioned above respectively, which is also consistent with analyses by SEM. Besides, we found that the interface of Ag and TiN layers is not very sharp, most likely because no shutter is provided to periodically shelter from Ag or TiN targets during multilayer deposition. On the other hand, diffusion phenomena can easily occur at the interfaces due to the higher energy of Ag+ and Ti+ during the deposition using a multi-arc ion plating technique. The XPS depth profiles also confirm that the mixed zone is existed between TiN and Ag, as shown in Fig. 2B, because the Ag not merely exists in Ag film but exists in TiN film in the TiN/Ag multilayer. In Fig. 2B, the XPS depth profiles also show the Ag atoms on the top TiN-Ag layer are approximately 3%, and the evident diffusion layers occur when the thickness of TiN-Ag film reaches 100 nm (Ag concentrations increase along with the diffusion amount of silver diffusion). From the periodic change of Ag, the multilayer structure of TiN/ 336
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Fig. 2. (A) SEM images (SE and BSE) of TiN/Ag nano-multilayer; (B) XPS depth profiles of Top TiN-Ag on TiN/Ag; (C) the XPS survey spectra; (D) high resolution Ag3d XPS spectra of different top layers. Table 1 The relative atomic percentage of Ag for different top layers. Groups
Samples
Ag atm.%
Test group 1 Test group 2 Test group 3
Top TiN on TiN/Ag Top Ag on TiN/Ag Top TiN-Ag on TiN/Ag
0.31 27.6 2.72
Notes: Measured by XPS. Abbreviations: Top TiN on TiN/Ag: the top TiN layer on TiN/Ag multilayer; Top Ag on TiN/Ag: the top Ag layer on TiN/Ag multilayer; Top TiN-Ag on TiN/Ag: the top TiN-Ag layer on TiN/Ag multilayer.
Ag multilayer is observed, and the modulation period is about 60 nm which is consistent with analyses by SEM. Meanwhile, the elemental composition and chemical states of different samples’ surfaces were analyzed by XPS, as shown in Fig. 2C and D. Ti, N and Ag are detected for all top layers as shown in Fig. 2C, which means the mixed zones are found on the surfaces of all the samples. The high-resolution Ag3d spectrum in Fig. 2D indicates that the Ag3d3/2 and Ag3d5/2 peaks are located at approximately 373.8 eV and 367.9 eV, respectively [32,33]. The two peaks of the Ag3d doublet correspond to metallic silver. Therefore, the Ag existed in the form of metallic silver in top Ag on TiN/ Ag and top TiN-Ag on TiN/Ag. And Table 1 gives the relative atomic percentage of Ag. According to the data presented in Fig. 2C–D and Table 1, the intensity of the two Ag3d doublet peaks increases with increasing atomic fraction of Ag. Fig. 3 shows the XRD patterns of the multilayers with three different top layers. All samples display strong a preferred orientation of TiN
Fig. 3. XRD patterns of different top layers.
(1 1 1), a weaker TiN(2 0 0) and TiN(2 2 0). Additionally, they also exhibit the signature of polycrystalline Ag(1 1 1). All the samples show the crystallographic orientations of TiN and Ag, because the top layers are so thin (∼120 nm for top TiN-Ag on TiN/Ag and top TiN on TiN/Ag, ∼10 nm for top Ag on TiN/Ag) that XRD provide the information of TiN/Ag multilayers. And the mixed zones are existed in the top layers confirmed by XPS. Fig. 4A–C show the load-unload cycles of the hardness tests on three samples. The indenter displacement in each figure is more than 200 nm, which means the hardness is attributed to top layer and the TiN/Ag
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Fig. 4. The load-unload cycles of (A) Top TiN layer on TiN/Ag; (B) top Ag layer on TiN/Ag; (C) top TiN-Ag layer on TiN/Ag; (D) the hardness, elastic modulus of different top layers.
presents the highest roughness values of Ra = 8.59 nm, Rq = 13.1 nm, Rz = 6.43 nm and Rt = 123 nm. While the roughness of the top TiN-Ag layers shows the lowest values of Ra = 0.687 nm Rq = 1.22 nm, Rz = 1.31 nm and Rt = 33.7 nm. This is probably because the Ag content of TiN-Ag composite layers can reduce the average crystallite dimension of TiN. The smaller grain size gives rise to a positive effect on the mechanical, frictional, and wear properties. So, in the friction coefficients of different top layers as shown in Fig. 6A, the top TiN-Ag layer exhibits the lowest the friction coefficient of 0.143, which is due to its smooth surface. Therefore, an appropriate Ag concentration on the surface can act as a lubricant which may effectively reduce the roughness and the friction coefficient, thereby improving the wear resistance of the surface. Fig. 6B shows the amount of Ag ions released from the three kinds of samples measured by ICP-AES. There are almost no Ag ions leached from top TiN on TiN/Ag, which may be due to the Ag+ concentration below the limit of detection. And, with time, the contents of Ag ions released from top Ag on TiN/Ag and top TiN-Ag on TiN/Ag all decrease slightly. In addition, the cumulative Ag+ concentration leached from top TiN-Ag on TiN/Ag is always lower than that from top Ag on TiN/Ag as immersion time is extended from 7 to 28 days.
multilayers, and the differences reflect different aspects of the top layer. For the TiN top layer (∼120 nm), the maximum load is 12.92 mN, and the final displacement after complete unloading is about 75 nm, which means it is a hard-ceramic brittle coating. Ag is a soft metal, but the top layer is so thin (∼10 nm) that the hardness mainly reflected the information of the TiN/Ag multilayers. Therefore, the final displacement after complete unloading does not show significant increasing, although the hardness decreases. For the TiN-Ag top layer (∼120 nm), the hardness increases due to the composite structure, which is favor to inhibit the dislocation motion. The final displacement after complete unloading is close to the Ag top layer due to the toughness increasing with the Ag inclusions. Fig. 4D presents the hardness and elastic modulus values of all samples. The hardness and elastic modulus of the top Ag layer on TiN/Ag shows the lowest values (16.02 GPa and 216.2 GPa, respectively), because Ag is a soft material with a low natural hardness [34,35]. The hardness and elastic modulus of the top TiNAg layer on TiN/Ag reaches the maximum values of 30.29 GPa and 341.9 GPa, respectively. The main reason for this is that the TiN-Ag composite with the appropriate Ag content can prevent the growth of TiN columnar crystals and large particles and inhibits dislocation motion, thus generating fewer defects and resulting in improved compactness. The hardness enhancement may be attributed to suppression of dislocation movement in small grains and the narrow space between the nanocrystalline particles and amorphous phases [36,37]. The strong interfaces between nanocrystalline and amorphous phases can increase the cohesive energy of the interface boundaries, restraining the grain boundary sliding. The higher hardness can resist material deformation and damage, which is very important for the implant material. Fig. 5A–E show the 3D image and roughness, including the arithmetic mean of surface roughness (Ra), root mean square of surface roughness (Rq), ten-point height of irregularities (Rz) and maximum peak to valley height (Rt) of different top layers. The top TiN layers
3.2. In vitro cytocompatibility studies The cell initial adhesion and spreading activity on surface of samples are assayed by staining with TRITC Phalloidin (Yeasen) and Hoechst 33342 (Gene Copoeia) to visualize the F-actin cytoskeleton (red) and nuclear DNA (blue), respectively. In this paper, the MC3T3-E1 cells layers cultured for 24 h on the surface of different top were stained, and the results are shown in Fig. 7. The expressions of F-actin of MC3T3-E1 cells on the surfaces of the top TiN-Ag layer exhibits multipolar spindle morphologies with numerous filopodia and 338
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Fig. 5. 3D images of surface topography, roughness and friction coefficient of different top layers: (A) 3D images of surface topography; (B) the Ra roughness; (C) the Rq roughness; (D) the Rz roughness; (E) the Rt roughness.
After 24 h, the attached cells are observed by SEM, as shown in Fig. 9. The cells adhere tightly to the surfaces of all samples. The cells on the top TiN-Ag layer spread extensively and exhibit multipolar spindle morphologies with numerous filopodia and lamellipodia, which is consistent with the results obtained by the fluorescent assay. In addition, the cell concentrations on the top TiN-Ag layer (the low magnification SEM images) are much higher than those on the top TiN or top Ag layers, indicating that the TiN-Ag composite layer with the appropriate silver content does indeed promote the cell adhesion. When a biomaterial implanted into body, the implants initially contact with body fluids. As the most abundant protein in body fluids, BSA plays an important role in the process of cell adhesion and growth [34,35] Fig. 8B clearly shows that the top TiN-Ag layer displays the highest adsorption quantity compared to the top TiN or top Ag layers. The results for the proteins adsorption are consistent with the image of protein electrophoresis, which reflects the quantity of the remaining BSA in the protein solution after the protein electrophoresis. Therefore,
lamellipodia. The F-actin of MC3T3-E1 cells on the surface of the top Ag layer reveals a wizened structure and a small number of filopodia and lamellipodia. These results indicate that while Ag can enhance the initial adhesion and spreading activity of the MC3T3-E1 cells, excessive amounts of silver are toxic for the cells. The appropriate silver content is more favorable to the growth of MC3T3-E1 cells with no cytotoxicity, corresponding to the cell proliferation assay. The proliferation of the MC3T3-E1 cells on all top layers was assessed with cell counting Kit-8 (CCK-8, Genview) after incubation for 24 h, 48 h and 72 h. The histogram of proliferation is shown in Fig. 8A. It is clearly seen that the cell number for the top TiN-Ag layer increases gradually from 24 h to 72 h, showing the highest cell proliferation. However, by contrast, the OD values of the top TiN and top Ag layers gradually decrease. This result shows that TiN-Ag as a top layer promotes the cell proliferation. Therefore, the TiN-Ag composite layer with the appropriate silver content accelerates the cell proliferation and growth.
Fig. 6. (A) The friction coefficient; (B) Ag concentrations in 0.9% NaCl solution after immersion for 7, 14, 21, and 28 days. 339
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Top Ag on TiN/Ag
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Fig. 7. Confocal laser scanning microscope images of MC3T3-E1 cells adhered on different top layers: (A–C) Top TiN layer; (D–F) top Ag layer; (G–I) top TiN-Ag layer.
Fig. 8. (A) The OD values of cells on different top layers versus incubation time; (B) the protein adsorption of different top layers.
TiN-Ag layers are ∼82%, ∼89.9%, ∼99.88%, respectively. The antibacterial ability of the concerned groups is ranked as follows: Top TiNAg on TiN/Ag > Top Ag on TiN/Ag > Top TiN on TiN/Ag. These results indicate that the antibacterial property of the samples mainly depends on the surface’s metallic silver, combined with the results of released silver ions test. The smaller reduction value of E. coli for the top Ag layer than that for the top TiN-Ag layer is attributed to the content of Ag exceeding a certain range, which reduces its antibacterial property. Marques’s experiment also obtained similar results [39]. The trace silver distributed in the antibacterial materials can activate the oxygen in the air or water and produce OH− and O2−. These active groups can react with bacteria and a variety of organic compounds, which can cause the fracture of the diacetyl bond between the bases of
the top TiN-Ag layers demonstrated to show better cytocompatibility than the other top layers. The cell adhesion, spreading activity and proliferation experiments all reach the same conclusion.
3.3. Antibacterial assay To evaluate the antibacterial activities of different samples against Escherichia coli (E. coli) in the first 24 h, the spread plate method was used. The adhered bacteria were detached from the samples and recultured on agar plates in accordance with the bacteria counting methods [38]. The results are shown in Fig. 10. The amount of viable E. coli on the top TiN-Ag layers the lowest among the three different top layers. The reduction values of E. coli on the top TiN, top Ag, and top 340
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pseudopod pseudopod MC3T3-E1 cell pseudopod
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Fig. 9. SEM images of MC3T3-E1 cells fixed on different top layers.
Fig. 10. (A) Re-cultivated E. coli colonies on agar plates of different top layers; (B) antibacterial rates of different top layers after being incubated for 24 h.
DNA chain. Thus, the process of metabolism is disturbed and the bacteria is killed. So the Ag content is a key parameter that affects the biocompatibility and antibacterial activity of the samples. Therefore, in this paper the top TiN-Ag layer on TiN/Ag not only promotes cell proliferation but also inhibits bacteria adsorption.
grain size should be a main reason for the best biocompatibility and antibacterial activity of the top TiN-Ag composite layer. At the same time, the Ag content is a key parameter that affects the biocompatibility and antibacterial activity of the samples. Therefore, an appropriate Ag content not only promotes cell proliferation but also inhibits bacteria adsorption.
4. Conclusion
Acknowledgements
In this paper, the top TiN, top Ag, and top TiN-Ag layers on TiN/Ag are deposited on medical titanium alloys using a multi-arc ion plating system. The top TiN or top Ag layers on TiN/Ag show a certain antibacterial property but inhibit cell proliferation and growth. However, the top TiN-Ag layer on TiN/Ag shows the lowest roughness, lowest friction coefficients, lowest contact angle, and the highest hardness and elastic modulus values, leading to the best cell adhesion, spreading activity and proliferation, as well as antibacterial activities. The smaller
This work was supported by National Natural Science Foundation of China (51772209) and Program for Innovative Research in University of Tianjin (Grant No. TD13-5077). References [1] H.J. Rack, J.I. Qazi, Titanium alloys for biomedical applications, Mater. Sci. Eng. C
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