Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys

Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys

Accepted Manuscript Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloy...

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Accepted Manuscript Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys

Sai Guo, Yanjin Lu, Songquan Wu, Lingling Liu, Mengjiao He, Chaoqian Zhao, Yiliang Gan, Junjie Lin, Jiasi Luo, Xiongcheng Xu, Jinxin Lin PII: DOI: Reference:

S0928-4931(16)30936-5 doi: 10.1016/j.msec.2016.11.126 MSC 7180

To appear in:

Materials Science & Engineering C

Received date: Accepted date:

22 August 2016 27 November 2016

Please cite this article as: Sai Guo, Yanjin Lu, Songquan Wu, Lingling Liu, Mengjiao He, Chaoqian Zhao, Yiliang Gan, Junjie Lin, Jiasi Luo, Xiongcheng Xu, Jinxin Lin , Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2016), doi: 10.1016/j.msec.2016.11.126

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ACCEPTED MANUSCRIPT Preliminary Study on the Corrosion Resistance, Antibacterial Activity and Cytotoxicity of Selective-Laser-melted Ti6Al4V-xCu Alloys

Sai Guo a,b, Yanjin Lu a, Songquan Wu a, Lingling Liu c, Mengjiao He c, Chaoqian Zhao a, Yiliang

a

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Gan a, Junjie Lin a, Jiasi Luo a,b, Xiongcheng Xu c, Jinxin Lin a,b,

Key Laboratory of Optoelectronic Materials Chemistry and Physics, Fujian Institute of Research

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University of Chinese Academy of Sciences, Beijing 100049, China

Key Laboratory of Stomatology (Fujian Medical University), Fujian Province University,

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c

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on the Structure of Matter, Chinese Academy of Sciences, China

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Fuzhou, Fujian, China

ABSTRACT

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In this study, a series of Cu-bearing Ti6Al4V-xCu (x=0, 2, 4, 6 wt.%) alloys (shorten by Ti6Al4V, 2C, 4C, and 6C, respectively.) with antibacterial function were successfully fabricated by selective

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laser melting (SLM) technology with mixed spherical powders of Cu and Ti6Al4V for the first

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time. In order to systematically investigate the effects of Cu content on the microstructure, phase constitution, corrosion resistance, antibacterial properties and cytotoxicity of SLMed Ti6Al4V-xCu alloys, experiments including XRD, SEM-EDS, electrochemical measurements,

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antibacterial tests and cytotoxicity tests were conducted with comparison to SLMed Ti6Al4V alloy (Ti6Al4V). Microstructural observations revealed that Cu had completely fused into the Ti6Al4V

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alloys, and presented in the form of Ti2Cu phase at ambient temperature. With Cu content increase, the density of the alloy gradually decreased, and micropores were obviously found in the alloy. Electrochemical measurements showed that corrosion resistance of Cu-bearing alloys were stronger than Cu-free alloy. Antibacterial tests demonstrated that 4C and 6C alloys presented strong and stable antibacterial property against Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus) compared to the Ti6Al4V and 2C alloy. In addition, similar to the Ti6Al4V 1

Corresponding author at: 155 West Yangqiao Road, Fuzhou, Fujian, the People's Republic of China, 350002 (Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences). Tel.: 86-591-83833016; Fax: 86-591-83833016. E-mail address: [email protected] (J.X. Lin).

ACCEPTED MANUSCRIPT alloy, the Cu-bearing alloys also exerted good cytocompatibility to the Bone Marrow Stromal Cells (BMSCs) from Sprague Dawley (SD) rats. Based on those results, the preliminary study verified that it was feasible to fabricated antibacterial Ti6Al4V-xCu alloys direct by SLM processing mixed commercial Ti6Al4V and Cu powder. Keywords: Titanium Alloy; selective laser melting; antibacterial property; corrosion resistance;

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cytotoxicity.

1. Introduction

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In the last several decades, Ti6Al4V alloys have been extensively used in clinical field, especially applied for load-bearing orthopedic implants in the physiological environment including

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orthopedics and dentistry[1] owing to their excellent integrative properties, such as specific high strength, low elastic modulus, low density, enhanced corrosion resistance and superior

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biocompatibility[2][3][4]. Nevertheless, as a kind of bioinert materials, commercial available Ti6Al4V alloys do not exert bactericidal capability after implantation, bacterial infections

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associated with implants happen occasionally, which greatly affect their further application and

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development in clinical field. In orthopedic surgeries, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the main pathogenic microbes responsible for the implant-related infections, especially S. aureus that accounts for about 34% of the infections[5][6][7]. The

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orthopedic implant related infections not only increase the time required for wound healing, but also influence the effectiveness of implantation. Moreover, some serious infections may result in

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implant loosening and implantation failure, even amputation and death in extreme cases[8][9]. In last decades, in order to solve the clinical bacterial infections, antimicrobial activity was imparted to the surface by applying a coating containing antimicrobial agents such as Cu[ 10 ][ 11 ][ 12 ], Ag[12][ 13 ] etc. Surface modifications including chemical vapor deposition[14][15], ion implantation[11], plasma spraying[12] and physical vapor deposition (PVD)[16]were adopted to suppress bacteria and impede the formation of biofilm[17]on the surface of implants. However, due to the discrepancy in material characteristics between some coatings and metallic substrates, surface modified coatings normally presented poor adhesive strength. For example, antibacterial TiO2 deposition coating always fell off from the substrate

ACCEPTED MANUSCRIPT owing to the poor adhesive strength. Besides, another crucial factor which led to the failure of antibacterial coating was the thin coating. For the ion implantation, the thin antibacterial surface was normally vulnerable, once destroyed by some reasons, the antibacterial property would disappear. Moreover, surface modified coatings only released antibacterial agents for a limited period of time after implantation, which meant that these coatings might only prevent early

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post-surgical infections caused by surgical contamination. Thus, the durability of antibacterial coatings still needed to be further enhanced.

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Therefore, it is necessary to develop a metal material with antibacterial activity in the whole alloy rather than only on the surface. Previous studies had been reported that by adding proper amount of

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Ag[18][19][20][21]or Cu[6][9][20][21][22][23][24] element into metal matrix followed by proper heat treatment, biomedical alloys with strong and broad-spectrum antibacterial activity had been

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developed. It was reported by Zhang et al.[22] that the addition of copper into Ti matrix fabricated by powder metallurgy provided the whole alloy with strong antibacterial property without

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reduction in mechanical property and corrosion resistance. Zheng et al.[25] reported that Ag particles precipitation within the TiNi alloy matrix made TiNiAg alloy to be a functional

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biomaterial which combined antibacterial activity and shape memory effect. Apart from that,

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Ti-6Al-4V-Cu alloys[9][23], titanium-copper alloys[6][22][24], Ti-Ag alloys[18] also showed good antibacterial properties.

However, most of extensive studies on manufacturing technology for antibacterial Ti alloys focus

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on the traditional processing methods, such as sintering[24], powder metallurgy[23] and casting[26], which not only need to prepare complex molds or machine tools, but also confront with

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formidable challenges, such as the oxidation problem of Ti6Al4V, phase transitions, decomposition and grain growth, etc[27]. Recent research efforts have demonstrated that SLM, due to its lower cost and flexibility in feedstock and shapes[28], possesses a promising potential for manufacturing antibacterial Ti6Al4V-xCu alloy parts directly from commercial Ti6Al4V powder and copper powder. Giving that, SLM provides a new approach to solve the serious problem of implants related bacterial infections via developing a novel class of metallic biomaterials with self-antibacterial function from the design of chemical composition for the implant materials, which means the problem of falling off from the matrix for the antibacterial coatings and problems in traditional processing methods mentioned above can be avoided.

ACCEPTED MANUSCRIPT To the authors’ best knowledges, there is little information concerning about the fabrication of Cu-bearing Ti6Al4V alloys by SLM process, which suggests that this study is of significant initiative and application value for practical application of Ti6Al4V-xCu alloys in orthopedics and dentistry. Therefore, in this study, the Cu-bearing Ti6Al4V alloys were attempted to be fabricated by SLM using mixture powders consisting of Cu and Ti6Al4V facilely. The effects of Cu content

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(2, 4, 6 wt.%) on the microstructure, corrosion resistance, antibacterial property, and cytocompatibility of SLMed Ti6Al4V-xCu alloys were systematically investigated for clinical

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application.

ACCEPTED MANUSCRIPT 2. Experimental 2.1 Preparation of SLMed Ti6Al4V-xCu alloys The Ti6Al4V-xCu (x=0, 2, 4, 6 wt.%) alloys (Unless particularly stated, the Ti6Al4V-xCu alloys below are referred to as SLMed alloys) were prepared from commercial Ti6Al4V powder and commercial pure Cu powder in selective laser melting processing by cross-hatching technique on

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a SLM machine (Mlab-R, CONCEPTLAER, Germany)[29][30]. After that, the alloys were water quenched. The average chemical composition of Ti6Al4V powder is listed in Table 1.

Chemical composition(wt.%) V 3.5-4.5

C 0-0.08

Fe 0-0.25

O 0-0.13

N 0-0.05

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Al 5.5-6.5

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Table 1. The Chemical compositon of Ti6Al4V powder

H 0-0.012

Ti Balance

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Before experiment, the samples were ground by using SiC grinding papers with 360, 1000 and 2000 grit sizes in turn under running water, then polished by using a SiO2-H2O2 solution, cleaned

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in acetone and alcohol by ultrasonic for about 30 min, and finally dried at room temperature for preparation.

2.2 Microstructural observation and Phase identification

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For phase identification and microstructure observations, samples were etched in Kroll reagent

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(containing 50 ml distilled water, 5ml HNO3 and 2ml HF) via a standard metallographic procedure. Microstructural observation was carried out on optical microscope (Axio Vert. A1, ZEISS) and scanning electron microscope (Phenom G2). An X-ray diffractometer (XRD, D/MAX-2500PC)

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with Cu Kα radiation was employed to identify the phase constitution of the experimental Ti6Al4V-xCu alloys. And the chemical compositions of the resulting Ti6Al4V-xCu alloys were

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detected by energy dispersive spectrum (EDS, Hitachi SU8010 SEM, Japan). The density of the samples with the dimension of 10×10×10 mm was measured on a precision balance (Shimadzu, TX2202L). 2.3 Electrochemical test The Electrochemical test of Ti6Al4V-xCu alloys was conducted to evaluate the corrosion resistance, which was conducted on a standard three-electrode configuration. Thereinto, a saturated calomel electrode (SCE) was used as the reference electrode, the counter electrode (CE) was a platinum electrode, and the samples with an exposed area of 0.64 cm2 were employed as the

ACCEPTED MANUSCRIPT working electrode (WE). All the three electrodes were immersed in 500 ml freshly 0.9% NaCl solution with the pH adjusted to be at 7.2-7.4 in a quartz corrosion cell, and the temperature was stabilized at 37 ± 1 °C by an electric-heated thermostatic water bath. The assembled corrosion cell was connected to an electrochemical working station (Gamry REFERENCE 600+, USA) controlled by a personal computer and dedicated Software.

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According to ISO 10271:2001 Standard[31], The open circuit potential vs. time curve of each specimen was continuously monitored for two hours in 0.9% NaCl solution to determine the

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open-circuit potential (EOCP). Afterward, the corrosion behavior for the passive film formed on the surface of specimens after 2 h immersion was studied by using electrochemical impedance

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spectroscopy (EIS) technique which was measured with a frequency range from 0.01 to 105 Hz. After that, the potentiodynamic polarization test were conducted in the potential range from -0.5

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V(SCE) up to 2.0 V with a constant scanning rate of 0.5mV/s. The tests were repeated at least three times for data reproducibility. The tangential lines of the anodic and cathodic curves were

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extrapolated to reach an intersection point to yield corrosion current density (Icorr), corrosion potential (Ecorr), and breakdown potential (Eb) following the standard approach, which called the

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Tafel extrapolation method. The corrosion rate (V) was calculated by [32]:

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V  MI/nF

(1)

Where, M is the molar mass of metal (g/mol), I is the average corrosion current density measured

Titanium.

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in the electrochemical tests (A/cm2), F is Faraday constant (96,485 C/mol) and n is the valence of

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In addition, every test was repeated three times for each material. All electrochemical data were analyzed using Gamry Instruments Echem Analyst version 7.03 software. 2.4 Immersion test

The static immersion test was performed in accordance with the currently specified ISO 10271:2001 standard for metallic biomaterials. All Ti6Al4V-xCu samples were fabricated by SLM with a size of 30×15×1.5mm (surface area>10cm2). Prior to performing this study, the samples were ground with waterproof emery paper to 2000 grits under running water, then ultrasonically cleaned in acetone, ethanol for 30min in turn, and finally dried at room temperature. The static immersion tests were conducted using lactic acid with a surface area/volume ratio of 1cm2/ml in

ACCEPTED MANUSCRIPT polypropylene bottles for 7 days at 37℃. Three specimens were tested for each group. Concentrations of metal ions released into solution were detected by an inductively coupled plasma atomic emission spectrometry (ICP-AES, Ultima2). 2.5 Antibacterial properties In this study, gram positive S. aureus, strain ATCC 6538 and gram negative E. coli, strain ATCC

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25922 were adopted to investigate the antibacterial activity of Ti6Al4V-xCu alloys. The Nutrient Agar (NA) for culturing the bacteria was prepared by dissolving 10.0 g peptone, 5.0 g beef extract,

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5.0 g NaCl and 20.0 g agar into 1000 ml of distilled water with pH value adjusted to 7.2-7.4. Before the microbiological experiment, all related instruments and samples were sterilized by an

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autoclave at 121 °C for 20 min.

Plate counting method was conducted with reference to Nation Standard of China GB/T

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2591-2003[33]for the assessment of the antibacterial property of Ti6Al4V-xCu alloys. Before the experiments, all glassware and samples were sterilized with UV irradiation for half an hour. For

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the antibacterial test, S. aureus and E. coli were cultivated at 37 °C in the nutrient agar to a concentration of around 108 cfu/ml, then were diluted 10-fold by PBS solution gradually to a

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concentration of 105 cfu/ml. After that, 50ul of the bacterial suspension with a concentration of 105

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cfu/ml was dripped onto the surfaces of each sample. Then, 24 well plate was incubated at 37 °C for 24 h under a humidity of 90%. After the incubation, the inoculated strain was harvested into a sterilized Petri dish by 2 ml sterilized physiological saline solution washing. The samples were

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carefully shed in order to make sure that no bacteria were left on the sample. Then, 50 ul of each bacterial suspension from above washing solution was inoculated onto nutrient agar plates and

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incubated at 37 °C for 24 h under a humidity of 90% before counting the surviving bacterial colonies. The active bacteria were counted in accordance with National Standard of China GB/T 4789.2-2010[34]. In order to obtain reliable and statistical results, three samples were assessed for each type of samples, where the Ti6Al4V alloys was used as negative comparison. The difference in the number of bacterial colonies for each sample was statistically analyzed. The antibacterial rate was calculated by the following formula:

Antibacterial rate (%)  (Ncontrol - Nsample )/N control 100%

(2)

where, Ncontrol and Nsample are the average numbers of the bacterial colony on the control samples

ACCEPTED MANUSCRIPT and the Ti6Al4V–Cu alloys, respectively. According to the National Standard of China (GB/T 4789.2-2010), R ≥ 90% means that the sample has antibacterial property, while R ≥ 99% indicates that the sample has strong antibacterial property. 2.6 Morphology of cells The preliminary cytotoxicity tests were carried out according to ISO 10993-5:2009[35]. The Bone

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Marrow Stromal Cells (BMSCs) harvested from Sprague Dawley (SD) rats were adopted to evaluate the cytotoxicity of Ti6Al4V-xCu alloys by direct cell assays. The BMSCs were cultured

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in Dulbecco’s modified eagle’s medium (DMEM, Hyclone, US) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified

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atmosphere with 5% CO2 at 37℃.

In the cell assay, the BMSCs with an initial density of 5×104 cells/ml were seeded on the sterilized

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Ti6Al4V-xCu samples in 24-well plates and then incubated for 1, 3 and 7 days, respectively at 37 ± 1 °C with 5% CO2 in a humidified incubator. The culture medium was refresh every other day.

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After each culture period, the specimens with cells were rinsed gently with phosphate buffered saline (PBS) for three times to remove non-adhered cells, and then fixed in 2.5% (w/v)

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glutaraldehyde for 1 h shielded from light. After this, the samples were dehydrated in gradient

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ethanol/distilled water mixture (50%, 60%, 70%, 80%, 90% and 100%) with interval of 10 min each, then dried in the air. Cell attachment and morphologies were observed with a Phenom G2 scanning electronic microscope.

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2.7 Cell viability assay

According to the International standard ISO 10993-12[36], Ti6Al4V-xCu alloys were immersed in

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DMEM solution with a surface area/volume ratio of 3 cm2/ml under a condition of 37 °C and 5% CO2 in air for 72 h to prepare extracts. The BMSCs were used to assess the cytotoxicity of Ti6Al4V-xCu alloys. Cell viability was determined with the CCK-8 cell viability assay (Dojindo Lab, Kumamoto, Japan) according to the manufacturer's protocol. During the procedure, 100 μl BMSCs suspension with a density of 5 × 104 cells/ml was pipetted in 96-well plates and cultivated at 37 °C with 5% CO2 in air for 24 h to allow attachment. Then, 10 μl of the extracts or medium was added into each well. The plate was then cultivated at 37 °C with 5% CO 2 in air for 1 day and 3 days. At the end of each incubation time, 20 μl CCK-8 solution was added into each well. After 2 h incubation at 37 °C with 5% CO2 in air, the optical density

ACCEPTED MANUSCRIPT (OD) of each well was measured with a Bio-Rad microplate reader at 450 nm. Five replicates were used for each sample to obtain a mean value. The cell viability was expressed by a relatively growth rate (RGR), which was calculated as the following equation:

RGR  (ODsample - ODblank ) /(ODnegativecontrol - ODblank ) 100% where, ODsample, ODnegative

control

(3)

and ODblank are the optical density of the Ti6Al4V and

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Ti6Al4V-xCu samples, the negative control (cells without extract) and blank control sample (no cells), respectively.

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2.8 Statistical analysis

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In all quantitative determinations, triple estimates were used. All numeric data were expressed as mean and standard deviations. The SPSS15.0 statistical software package was used for data

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analysis, where, P>0.05 was considered as no statistically significant difference.

ACCEPTED MANUSCRIPT 3. Results 3.1 Phases and Microstructure Fig.1 presented the XRD patterns of Ti6Al4V-xCu alloys obtained at room temperature. In Ti6Al4V alloys, it could be found that the diffraction peaks of typical α and β phase were detected. With the Cu addition, the diffraction peaks of Ti2Cu intermetallic phase were also observed apart

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from the diffraction peaks of the Ti6Al4V matrix. Moreover, it was noted that the relative diffraction intensity of Ti2Cu phase gradually increased with increasing the Cu contents, indicating

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that more Ti2Cu phase were synthesized. However, there was no metallic Cu was detected in all alloys, which indicated that Cu had fused into the Ti6Al4V alloy existing in form of Ti2Cu phase

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after the SLM processing.

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Fig. 1 XRD patterns of Ti6Al4V-xCu alloys.

Fig. 2 Density and porosity of Ti6Al4V-xCu alloys. Fig. 2 illustrated the density and porosity of Ti6Al4V-xCu alloys. With increasing the addition of Cu, the discrepancy between theoretical density and actual density became evident, indicating ever-increasing porosity in the alloys, which rose to 3.78% in 6C alloy. Fig.3 and Fig.4 showed the representative surface of Ti6Al4V and Cu-bearing Ti6Al4V alloys at a low and high magnification,

ACCEPTED MANUSCRIPT respectively. In the low magnification images (Fig. 3), micropores scattered randomly on the matrix of Ti6Al4V-xCu alloys, especially for 6C sample, indicating that Cu addition in Ti6Al4V alloys might cause structural defects to some extent. The observation was consistent with the results presented in Fig. 2. In the high magnification images (Fig. 4), it was clear to see that these pores were independent and mutually disconnected, and most of them presented as near-spherical

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morphology with a size of 10-20 μm. As for the change of metal phases (in Fig.3), the typical chessboard pattern in Ti6Al4V alloy

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disappeared after the Cu addition in the alloys. Whilst many rod-like α phase and laminar β phase anomalously distributed in the matrix of Ti6Al4V-xCu alloys. With increasing Cu content, the

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rod-like α phase became short and sparse and its amount decreased, while the amount of β phase and the size increased. The change could be clearly observed in Fig.4. Fig.5 presented the

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elemental content in α and β phase obtained from 6C alloy (in Fig.4d) by EDS. Obviously, as a β phase stabilizer in titanium alloys, Cu inclined to preferentially exist in β phase rather than α phase,

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as a result, higher content of Cu was detected in β phase than that in α phase.

Fig. 3 Microstructure of Ti6Al4V-xCu alloys with a magnification of 100 times under an optical microscope. (a) Ti6Al4V, (b) 2C, (c) 4C, and (d) 6C.

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Fig. 4 Microstructure of Ti6Al4V-xCu alloys under a SEM.

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(a) Ti6Al4V, (b) 2C, (c) 4C, and (d) 6C.

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Fig. 5 Comparison of elemental content in α and β phase obtained from 6C alloy by EDS. *p < 0.05.

3.2 Electrochemical tests The impedance spectra of Ti6Al4V-xCu alloys immersed in 0.9% NaCl solution at 37℃ were represented as Nyquist plots (Fig. 6a) and Bode diagrams. In Nyquist plots, all alloys represented as a semi-circular arc dependence of the real, Zreal, and imaginary, Zimag, components, which were characterized as EIS typical mono-layer film. The moduli of impedance (Z) was characterized by large semicircle and larger value of impedance suggested higher corrosion resistance. As presented in Fig. 6a, with the increase of Cu concentration, the diameter of semi-circular of the Nyquist loop increased to the maximum with an addition of 4 wt.% Cu, displaying that the formation of a dense

ACCEPTED MANUSCRIPT passive layer on the metal surface improved the corrosion resistance. Then the diameters gradually depressed with further increase of Cu content, indicative of the formation of a porous passive layer on the metal surface of 6C alloy corresponding to poorer corrosion resistance. In spite of that, the diameter of 6C alloy still was larger than the Cu-free Ti6Al4V alloy. Bode plots obtained from Ti6Al4V and the Cu-containing alloys were quite similar, including the

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frequency vs. impedance magnitude |Z| (Fig. 6b) and the frequency vs. phase angle curves (Fig 6c). Where, the Ti6Al4V alloy exhibited a lower phase angle and impedance value (Fig. 6 b and c),

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indicating a less resistant passive oxide film than that present on the copper-bearing Ti6Al4V alloys surface. As could be seen from Fig. 6b, the 4C alloy possessed the highest phase angle of

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68.3˚, much higher than Ti6Al4V with a phase angle of 58.6˚ in a fixed 0.01 Hz frequency, indicative of a highly stable barrier layer of passive film in 4C alloy. As presented in Fig. 6b, the

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spectrum in the middle frequency range (0.1-1000Hz) exhibited a broad linear region with a slope of about -1, which was the typical feature of mono-layer passive layer. And the log |Z| vs. log f

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curve of Cu-bearing alloys displayed greater inclination than that of Ti6Al4V alloy, denoting a higher barrier layer capacitance. In addition, considering the frequency vs. phase angle curves in

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Fig. 6c, Ti6Al4V and its Cu-containing alloys had only one time constant element. Thus, the

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equivalent electrical circuit (EEC) model (in Fig. 7) with one constant phase element (CPE) was used to evaluate the impedance spectra in the situations of single passive layer–electrolyte interface. The admittance and impedance of CPE were expressed below, respectively:

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YCPE  Y0 ( j)n

(5)

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ZCPE  (1 / Y0 )( j)  n

(4)

where Y0 is the magnitude of the CPE, j the imaginary number or (-1)1/2, ω the angular frequency, and n the CPE power index which is less than unity[37]. Thereinto, Rs and Rp correspond to the resistances of the electrolyte (0.9% NaCl solution) and the passive layer, respectively. Q1 represents the capacitance of the layer. The decreasing of n value from 1 displays the increasing of the porosity in the passive layer. In other words, the passive layer becomes more dense as the n value is close to n = 1[38]. Table 2 listed the electrochemical parameters obtained from the impedance data analysis software. In the experiments, with the increase of Cu content, the Rp of Ti6Al4V-xCu alloys increased with

ACCEPTED MANUSCRIPT 4 wt.% Cu addition at first then took on a ever-decreasing trend to 6C, indicating that a slight Cu addition in Ti6Al4V alloy helped strengthen its corrosion resistance. Nevertheless, overmuch Cu addition exerted a adverse impact on the corrosion resistance, but still better than the Ti6Al4V alloy overall. Furthermore, the impedance and the phase angle at the intermediate frequency and low frequency range decreased with the increase of Cu content over 4 wt.% addition, indicating

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the formation of porous passive layers with poorer corrosion resistance. Where, 4C alloy exhibited the highest corrosion resistance, much higher than that of the Ti6Al4V alloy.

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The potentiodynamic polarization curves of all samples in 0.9% NaCl solution at 37℃ were presented in Fig. 6d. The corrosion current density (Icorr), corrosion potential (Ecorr), and

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breakdown potential (Eb) yielded from the Tafel extrapolation method were summarized in Table 3. It was noteworthy that all polarization curves of Ti6Al4V-xCu alloys were quite similar.

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Compared with the curve of Ti6Al4V alloy, the curves of Cu-bearing Ti6Al4V alloys shifted upward leading to an increase of Ecorr and decrease of Icorr. With increasing Cu content, the Icorr

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value decreased to 12.9 nA of 2C from 58.6 nA of Ti6Al4V alloy, indicating the corrosion resistance was enhanced by the addition of Cu. And more increase of Cu led to a further decline of

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Icorr value to 10.0 nA of 4C. Nevertheless, 6 wt.% Cu addition in Ti6Al4V alloy caused a rise of

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Icorr value to 18.2 nA, suggesting the decline of corrosion resistance. However, the Icorr value of the 6C alloy was still more noble than that of the Ti6Al4V alloy. Apart from that, the Eb value exhibited no significant difference with increasing Cu in Ti6Al4V alloy, while Ti6Al4V displayed

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a broader passivation region than the Cu-containing alloys. Where, the passivation region of Ti6Al4V ranged from 46.2 mV to 101.8 mV, broader than that of 4C from 392.9 mV to 101.4 mV.

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However, Cu-containing alloys possessed higher initial passivation potential, indicating the formation of thicker passive layer with Cu addition. In general, The impact of Cu content on the corrosion resistance of Ti6Al4V-xCu alloys from the potentiodynamic polarization plots were consistent with the EIS tests.

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Fig. 6 (a) Nyquist plots; (b) Frequency vs. impedance magnitude |Z| curves; (c) Frequency vs.

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phase angle curves; (d) Potentialdynamic polarization curves of Ti4Al4V alloys with different Cu contents.

Rp×106 (Ω∙cm-2)

Yo ( S∙sn ∙cm-2)

n

3.22± 0.15 4.08± 0.22 2.08±0.68 8.20±0.21

2.50E-05 2.32E-05 2.43E-05 4.60E-05

0.909 0.912 0.899 0.803

44.73±0.28 38.02±0.24 41.23±0.26 33.66±0.21

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2C 4C 6C Ti6Al4V

Rs (Ω∙cm-2)

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Samples

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Table 2. Electrochemical data of Ti4Al4V alloys with different Cu contents from EIS tests.

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Table 3. Electrochemical data of Ti4Al4V alloys with different Cu contents from OCP curves and potential-dynamic polarization curves .

Samples

Icorr (nA)

Ecorr (mV)

Eb (mV)

Corrosion rate(mg∙cm-2∙yr-1)

2C 4C 6C Ti6Al4V

12.9 10.0 18.2 58.6

-500 -453 -447 -536

101.7 101.4 101.6 101.8

0.154 0.119 0.217 0.669

ACCEPTED MANUSCRIPT Fig. 7 The typical ECD (Equivalent circuit diagram) for fitting the impedance spectra of Ti6Al4V-xCu alloys in 0.9% NaCl solution. 3.4 Metal ion release Table 4 presented the accumulative ion concentrations of Cu released from Ti4Al4V alloys with different Cu contents after immersion in lactic acid solution for 7 days. With the increase of Cu

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content in Ti6Al4V-xCu alloys, Cu ion exhibited a significant increase, and the ion concentration corresponded to its relative content in corresponding alloy. The calculated Cu ion release rates was

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also listed in the table.

Table 4. Cu ion concentration and Cu ion release rate in lactic acid solution for 7 days. Cu ion concentrations, mg/L

Cu ion release rate, mg/cm2/d

Ti6Al4V

-*

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2C

0.385

4C

0.585

6C

0.773

5.5×10-5

8.36×10-5 1.10×10-4

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*- means undetected. 3.5 Antibacterial activity

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Fig. 8 presented the E. coli colonies incubated for 24 h on Ti6Al4V alloy (the control sample) and

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Cu-bearing Ti6Al4V alloys. It could be seen that large amount of bacterial colonies were observed on the Ti6Al4V sample, which confirmed the fact that Ti6Al4V alloy did not have antibacterial

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activity. Similarly, a large number of bacterial colonies were also observed on 2C sample, displaying a weak antibacterial activity. However, when Cu content mounted to 4 wt.%, only a

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few bacteria colonies were found. and when the Cu content reached 6 wt.%, nearly no bacterial colony was found on the plate, indicating that 4C and 6C samples had strong antibacterial activity against E.coli. Similar results were also found in the antibacterial experiment against S. aureus as presented in Fig. 9: lots of bacteria were on the control sample as well as 2C sample, but only a few bacteria were found on 4C and 6C samples.. The change of the calculated antibacterial rate with different Cu contents was presented in Fig. 10. The average antibacterial rate of 2C against E. coli and S. aureus were 51.54% and 60.94%, respectively, less than 90%, indicating the alloy did not have antibacterial activity according to Standard (GB/T 4789.2-2010). It had to be pointed out that the standard deviations were very high,

ACCEPTED MANUSCRIPT displaying that the antibacterial activity of this alloy was not stable. When the Cu content rose to 4 wt.%, the antibacterial rate sharply soared to a very high value, as high as 97.40% against E. coli and 93.56% against S. aureus, respectively, meaning that this alloy had antibacterial property. When Cu content mounted to 6 wt.%, the antibacterial rate were larger than 99%, meaning that these alloys had strong antibacterial activity. It could be deduced from the above results that the

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Cu content had a significant influence on the antibacterial property and the Cu content in Ti6Al4V-xCu alloys had to be at least 4 wt.% in order to get a strong and stable antibacterial

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activity.

Fig. 8 E. coli bacterial colonies after incubation for 24 h on different samples,

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(a) Ti6Al4V sample, (b)2C sample, (c)4C sample, (d)6C sample.

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Fig. 9 S. aureus bacterial colonies after incubation for 24 h on different samples,

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(a) Ti6Al4V sample, (b)2C sample, (c)4C sample, (d)6C sample.

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Fig. 10 Antibacterial rate of Ti6Al4V-xCu alloys against E. coli and S. aureus with change in Cu content.

3.6 Morphology of cells Figs. 11 showed the morphology of BMSCs cultivated on the samples for 1day, 3days and 7 days, respectively. After 1 day incubation, there was a confluent layer of cells attached on the surfaces of all Ti6Al4V-xCu samples. No difference could be found in the cell morphology, indicating that the Cu addition did not bring any ditrimental impact to the cell morphology after 1 day cultivation. Also, the interactions among cells were observed with the presence of filopodia extending from the cells. With the increase of culture time, the cells became denser and spread better, more

ACCEPTED MANUSCRIPT lamellipodia could be observed both on the Ti6Al4V and Cu-containing samples after 3 and 7 days incubation, as shown in Fig. 11. There was no significant difference on quantity and morphology of these four groups, which indicated that Cu addition up to 6 wt.% in Ti6Al4V alloy

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had no negative effect on cell proliferation and activity.

Fig. 11 Morphology of BMSCs on Ti6Al4V-xCu samples after 1, 3 and 7 days incubation. (a), (e) and (i) Ti6Al4V; (b), (f) and (j) 2C; (c), (g) and (k) 4C; (d), (h) and (l) 6C.

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3.7 Cell viability assay

The influence of the Ti6Al4V-xCu alloys on the proliferative activity of BMSCs was assessed by

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CCK-8 test. Fig. 12 presented the calculated RGRs of BMSCs on Ti6Al4V-xCu alloys with different cultivation durations. After incubation for 1 day and 3days, all RGRs remained at a elevated level, larger than 90%. Besides, the statistical analysis presented no difference (p>0.05) in RGR values between negative control samples and Cu-bearing alloys at both cultivation points. According to the ISO-10993-5[35], the cytotoxicity of a biomedical material is ranked Grade 0 when RGR is over 90% which implies that the material has no cytotoxicity to the cell. In the cell viability assay, the cytotoxicity scale of all samples were ranked as Grade 0 at both day 1 and day 3, indicating that BMSCs grew at a high proliferation rate and Ti6Al4V-xCu alloys exhibited good cytocompatibility to BMSCs.

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Fig. 12 RGR values of Ti6Al4V-xCu alloys with incubation duration.

ACCEPTED MANUSCRIPT 4. Discussion Generally, alloying elements selected for developing antibacterial alloy should have antibacterial activity in nature as well as exert no negative influence on other properties of matrix alloy[22]. As an essential trace element, copper is of crucial importance to the proper functioning of organs and metabolic processes since it participates in the formation of red blood cells, the absorption and

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utilization of iron, the metabolism of cholesterol and glucose, and the synthesis and release of life-sustaining proteins and enzymes[39]. Moreover, Cu element has been confirmed to be a

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highly efficient antibacterial agent when alloyed with Ti[22][24], Ti6Al4V[9][23] and 317L stainless steel[40]. According to the equilibrium phase diagram of the Ti-Cu binary alloys, only a

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secondary phase, i.e., Ti2Cu phase forms when alloyed with less than 40 wt.% Cu under an equilibrium solidification condition[26][41]. In this study, the existence of Ti2Cu phase and

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undetected Cu phase by XRD in Ti6Al4V-xCu alloys indicated that Cu had completely fused and alloyed with Ti6Al4V alloy by mutual diffusion during SLM process. Besides, it could be seen

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from Fig. 4 and Fig. 5 that with increasing Cu content, the β/ɑ phase ratio increased gradually and the Cu tended to exist in β phase. This was due to Cu, a β phase stabilizer in titanium alloys, could

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reduce ɑ-β phase transition temperature, as consequent of promoting the transition process. Apart

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from the phase change of Ti6Al4V alloys with different Cu content, the density of Ti6Al4V-xCu alloys also had close relation with SLM process. In this study, the increase of Cu content caused the porosity in the matrix, as could be seen from Fig.3. According to Cunningham et al.[42], there

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are multiple types of porosity in additively manufactured materials in general, including lack of fusion porosity with large than 100 μm, trapped gas porosity with near-spherical morphology, and

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keyhole pores resulting from alloys’ vaporization[43]. In this study, as shown in SEM images (Fig. 4), lots of near-spherical pores with size around 10 μm randomly scattered on the surface of Ti6Al4V-xCu alloys. These pores were generally characterized with regular shape and small size(<100 μm), which were apparently identified as trapped gas porosity. Good corrosion resistance was one of the most significant properties for Ti6Al4V-xCu alloys to be applied as orthopedic and dental implants. In static immersion testing, results from the metal release revealed that ion concentration in solution mounted with the Cu content in Ti6Al4V-xCu alloys. It's worth noting that the maximum Cu ion release in lactic acid solution came from 6C alloys with a concentration of 0.773 mg/L/7days, i.e., the daily average release of Cu ion from the

ACCEPTED MANUSCRIPT alloy was 1.1 ug/cm2/day (11ug/day). The value was much lower than the recommended minimal acceptable intake of Cu for an adult by WHO (approximately 1.3 mg/day)[44]. Another assessment for corrosion resistance of Ti6Al4V-xCu alloys was conducted by electrochemical testing, the results demonstrated that comparing with the Ti6Al4V alloy, Cu-containing samples generally exhibited much better corrosion resistance, where 4C sample

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displayed the most outstanding corrosion resistance. In theory, with increasing Cu content, Ti6Al4V-xCu alloys should show nobler electrochemical behavior due to the addition of nobler Cu

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which helps to form the passive film with higher thermodynamical stability on the metallic surface in aqueous solutions[45][46]. In fact, the corrosion resistance and transpassive potential of

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Ti6Al4V alloyed with over 4 wt.% Cu took on a downward trend, instead. To explain the phenomenon, the porosity, amount of α and β phases and Ti2Cu intermetallic phases should be

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involved. On one hand, intermetallic phases are generally nobler when compared to the metallic matrix. Takahashi et al.[47]reported that intermetallic Ti3Au particles in Ti-Au alloys dissolved

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preferentially with an abrupt increase in the current density in a 0.9% NaCl solution, which acted as a protection against corrosion. Thus, similar to the intermetallic Ti3Au particles in Ti-Au alloys,

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it is reasonable to consider that the initial metal release may be attributed to the dissolution of

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intermetallic Ti2Cu phase on the surface until the formation of passive surface oxide, which could account for the improved corrosion resistance with more Ti2Cu phases in Ti6Al4V-xCu alloys. On the other hand, according to Ma’s research[9], Cu existed in two different states in Cu-bearing

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Ti6Al4V alloys, i.e., interstitial solid solution in the α and β phases, and precipitation of intermetallic Ti2Cu phase. Compared with the stable Ti2Cu phase, the presence as interstitial atoms

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was easier for the release of Cu ions from the alloys through spontaneous diffusion under the driving force provided by concentration gradient. Besides, the addition of Cu in Ti6Al4V alloys also brought negative influence to SLM process to a certain degree, just as discussed above, which resulted in micropores on the surface (Fig. 3 and 4). The micro-pores thereby enlarged surface area, with the consequent influences of causing crevice corrosion attack and localized breakdown of passive layer[48]. As analyzed above, it could be conclude that the porosity and Ti2Cu intermetallic phases could explain why the corrosion resistance decreased with the increase in Cu. However, it was also found that the Cu addition also enhanced the corrosion resistance of Ti6Al4V, although more pores were formed in the

ACCEPTED MANUSCRIPT Ti6Al4V-xCu alloys. This was because, for one thing, those trapped gas porosity were normally independence and not connected to each other, therefore, the detrimental influence on the corrosion resistance caused by those trapped gas porosity on the surface was limited relatively; for another, the increase of β phase with the addition of Cu should be responsible for the phenomenon. Therefore, there might be some relationship between the β/ɑ phase ratio and corrosion behavior of Ti6Al4V-xCu alloys. That is to say, the Ti6Al4V-xCu alloys with higher amount of β phase could

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further investigation should be conducted to verify the deduction.

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enhance the corrosion resistance compared with the lower amount of β phase. However, the

Besides that, the release behavior of Cu ion was closely related to the antibacterial activity for an

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antibacterial alloy. Many literatures demonstrated the controlling mechanism was not yet completely understood. Rodrigues et al.[48] pointed out that the main reason of antibacterial

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ability was the Cu ions release from materials that destroy the cell walls, which became thick and coarse, and then the cytoplasm degraded and disappeared, leading to cells death. Sterritt et al.[49]

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believed that the antibacterial mechanism was possibly resulted from the copper destructed enzyme structures and functions by binding to sulfur- or carboxylate-containing groups and amino

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groups of proteins. Therefore, it was widely accepted that Cu ion released from Cu-bearing

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material played a vital role in the antibacterial activity. In this study, it could be seen from Fig.10 that the Ti6Al4V-xCu alloys with 2wt.% Cu addition showed a poor antibacterial rate of lower than 60%, whereas Ti6Al4V-xCu alloys with 4 and 6 wt.% Cu addition exhibited relatively strong

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antibacterial activity against E.coli and S. aureus. Similar results had also been obtained by the other researchers. For example, Ren et al.[40] has deeply studied the antibacterial mechanism of

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the Cu-bearing stainless steel and confirmed that the dissolution of Cu ions from the Cu-SS played the key role of killing bacteria when it contacted with bacteria. From the results of metal release and antibacterial test, there was a distinctive relationship between the amount of Cu release and antibacterial ratio. That was, the more amount of release Cu, the stronger antibacterial activity. Wang et al.[50] reported that the precipitation of Cu-rich phase in the cast CoCrWCuNi alloy exert strong antibacterial performance via releasing the strong antibacterial agent of Cu ions. Zhang et al. who fabricated Cu-bearing antibacterial stainless steel found that more Cu ions were released from surface of the antibacterial stainless steel and exerted stronger antibacterial effect. According to the conclusion of Liu et al., only Ti-Cu alloys with more than 5 wt.% Cu would exhibited strong

ACCEPTED MANUSCRIPT antibacterial activity, proving again that the antibacterial activity was significantly dependent on the Cu content. From the antibacterial results, there was no doubt that the antibacterial activity was closely related to the Cu content. Apart from good antibacterial activity, a good biocompatibility with the surrounding tissue should be considered simultaneously before application of an implant biomaterial. Like all essential

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elements and nutrients, too much or too little nutritional ingestion of copper can result in a corresponding condition of copper excess or deficiency in the body, each of which has its own

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unique set of adverse health effects. Copper insufficiency is often associated with severe pathologic alterations including impairment of blood, liver, and immune systems[51]. However,

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excess copper intake causes stomach upset, nausea, and diarrhea and can lead to tissue injury and disease. Therefore, it was necessary to produce copper-containing biomaterials with proper Cu

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addition that suppress bacterial growth without being cytotoxic. In this study, the cytotoxicity test results showed that BMSCs exhibited a good cell proliferation and attachment on the surface of

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Ti6Al4V-xCu alloys. And the RGRs of BMSCs incubated in the extacts of Ti6Al4V-xCu alloys maintained at a relatively high level, which preliminarily testified that 6wt.% Cu addition in

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Ti6Al4V alloy exhibited an acceptable in vitro biocompatibility, which was similar to the previous

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results obtained by Ma et al.[9] who reported that electrode arc-melting Ti6Al4V alloy with 5 wt.% Cu element presented good cytocompatibility to MC3TC-E1 cells. As such, Zhang et al.[52] also pointed out that Ti powder sintered with even 25 wt.% Cu addition did not bring about any

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difference in the cell adhesion and viability of MG63 cell. To further assess the biocompatibility of the Ti6Al4V-xCu alloys, the in vivo and long term biocompatibility of Ti6Al4V-xCu alloys should

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be conducted.

In general, above results demonstrated that the Cu content in Ti6Al4V-xCu alloys seriously influenced the corrosion resistance and the Cu ion release behavior. Even though the micropores in the matrix resulting from the SLM process have adverse impact on the corrosion resistance, the Ti6Al4V alloys with 2, 4, 6 wt.% Cu addition generally shown much stronger corrosion resistance comparing with the Cu-free Ti6Al4V alloy. When Ti6Al4V alloys added with over 4 wt.% Cu exerted strong and stable antibacterial property against E.coli and S. aureus. Also, all Ti6Al4V-xCu alloys in this study exhibited good cytocompatibility. However, the mechanical property, in vivo antibacterial performance and biocompatibility of Ti6Al4V-xCu alloys still need

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further study to get comprehensive property for clinical application.

ACCEPTED MANUSCRIPT 5. Conclusion In this study, a set of antibacterial Ti6Al4V-xCu alloys was successfully fabricated by selective laser melting process. And the effects of Cu content (2, 4, 6 wt.%) on the microstructure, antibacterial property anticorrosion property and cytocompatibility of SLMed Ti6Al4V-xCu alloys had been systematically investigated. Within the limits of this study, the following conclusions

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were drawn: (1) The addition of Cu in the matrix of Ti6Al4V-xCu alloys during SLM process caused

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micropores in the matrix, which were mainly characterized as the trapped gas porosity. And the porosity and β phase mounted with increasing the Cu content in SLMed Ti6Al4V-xCu alloys.

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(2) The SLMed Ti6Al4V alloys with 2, 4, 6 wt.% Cu addition presented much stronger corrosion resistance comparing with the Ti6Al4V alloy. Thereinto, 4 wt.% Cu addition in Ti6Al4V alloys

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displayed the most outstanding corrosion resistance.

(3) The antibacterial activity was significantly dependent on the Cu content. Namely, the SLMed

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Ti6Al4V-xCu alloys with 4 and 6 wt.% Cu addition exhibited strong antibacterial property. (4) SLMed Ti6Al4V-xCu alloys showed good cytocompatibility to BMSCs.

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(5) The SLM antibacterial Ti6Al4V-xCu alloys direct fabricated from mixed commercial Ti6Al4V

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antibacterial implants.

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and Cu powder were verified to be feasible, which was a promising approach to obtain

ACCEPTED MANUSCRIPT Acknowledgment The authors would like to acknowledge the financial support by National projects (No. 2016YFC1100500, 2016YFC1102801 and XDA09020301). We also appreciate the corporation of

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the Key Laboratory of Stomatology of Fujian Medical University for providing biology laboratory.

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References

ACCEPTED MANUSCRIPT Highlights 

Direct SLM of Ti6Al4V-xCu alloys by mixing Ti6Al4V and a few Cu powder is feasible.



The trapped gas porosity exhibits a limited effect on the corrosion resistance.



SLMed Ti6Al4V alloys with 4 and 6 wt.% Cu exhibited stronger antibacterial property.



SLMed Ti6Al4V-Cu alloys show good cytocompatibility.

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[1] K. Wang, Mater. Sci. Eng. A 213 (1996) 134-137.

[2] M. Peters, J. Hemptenmacher, J. Kumpfert, C. Leyens, Structure and properties of titanium

RI

and titanium alloys, in:C. Leyens, M. Peters (Eds.), Titanium and Titanium Alloys:

SC

Fundamentals and Applications, Wiley-VCH, Weinheim, 2003.

[3] M. Koike1, Z. Cai, Y. Oda, M. Hattori, H. Fujii, T. Okabe, J. Biomed. Mater. Res. B 73

NU

(2005) 368-374.

[4] O.Z. Andersen, V. Offermanns, M. Sillassen, K.P. Almtoft, I.H. Andersen, S. Sørensen,

MA

Biomaterials 34 (2013) 5883-5890.

[5] C. Davide, M. Lucio, R.A. Carla, Biomaterials 27 (2006) 2331-2339.

91B (2009) 373-380.

D

[6] T. Shirai, H. Tsuchiya, T. Shimizu, K. Ohtani, Y. Zen, K. Tomita, J. Biomed.Mater. Res. B

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[7] E. Barth, Q.M. Myrvik,W.Wagner, A.G. Gristina, Biomaterials 10 (1989) 325–328. [8] H.Y. Liu, X.J. Wang, L.P. Wang, F.Y. Lei, X.F. Wang, H.J. Ai, Appl. Surf. Sci. 254 (2008) 6305–6312.

CE

[9] Z. Ma, L. Ren, R. Liu, K. Yang, Y. Zhang, Z.H. Liao, W.Q. Liu, M. Qi, R.D.K. Misra, J. Mater. Sci. Technol..31 (2015) 723-732.

AC

[10] F. Heidenau, W. Mittelmeier, R. Detsch, M. Haenle, F. Stenzel, G. Ziegler, H. Gollwitzer, J. Mater. Sci. Mater. Med. 16 (2005) 883–888. [11] Y.Z. Wan, G.Y. Xiong, H.Liang, S. Raman, F. He, Y. Huang, Appl. Surf. Sci. 253 (2007) 9426–9429. [12] Li B, Liu X, Meng F, Chang J, Ding C, Mater Chem Phys 2009;118:99-104. [13] K.D. Secinti,M. Ayten, G. Kahilogullari, G. Kaygusuz, H.C. Ugur, A. Attar, J. Clin. Neurosci. 15 (2008)434–439. [14] X. Bai, K. More, C.M. Rouleau, A. Rabiei, Acta Biomater. 6 (2010) 2264–2273. [15] U. Brohede, J. Forsgren, S. Roos, A. Mihranyan, H. Engqvist, M. Stromme, J. Mater. Sci.

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Mater. Med. 20 (2009) 1859–1867. [16] Ewald A, Glückermann SK, Thull R, Gbureck U, Biomed. Eng.Online 2006;5. [17] L.Z. Zhao, P.K. Chu, Y.M. Zhang, Z.F. Wu, J Biomed Mater Res 2009;91b:470-80. [

18] M.K. Kang, S.K. Moon, J.S. Kwon, K.M. Kim, K.N. Kim, Mater. Res. Bull. 47 (2012) 2952–2955.

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[19] Y. Oda, M. Funasaka, T. Sumii, J J Dent Mater 1990;9:314 –319. [20] M. Kikuchi, M. Takahashi ,T. Okabe, O. Okuno, Dent Mater J 2003;22:191-205.

RI

[21] M. Takahashi, M. Kikuchi, Y.Takada, O. Okuno, Dent Mater J 2002;21:270-80.

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[22] E.L. Zhang, F.B. Li, H.Y. Wang, J. Liu, C.M. Wang, M.Q. Li, K. Yang, Mater. Sci. Eng. C 33 (2013) 4280-4287.

[23] L. Ren, Z. Ma, M. Li, Y. Zhang, W.Q. Liu, Z.H. Liao, K. Yang, J. Mater. Sci. Technol., 2014,

NU

30(7), 699-705.

[24] J. Liu, F.B. Li, C. Liu, H.Y. Wang, B.R. Ren, K. Yang, E.L. Zhang, Mater. Sci. Eng. C 35

MA

(2014) 392-400.

[25] Y.F. Zheng, B.B. Zhang, B.L. Wang, Y.B. Wang, L. Li, Q.B. Yang , L.S. Cui. Acta Biomaterialia 7 (2011) 2758–2767.

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Mater 19 (2003) 174-181.

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[26] M. Kikuchi, Y. Takada, S. Kiyosue, M. Yoda, M. Woldu, Z. Cai, O. Okuno, T. Okabe, Dent

[27] G. Lutjering, J.C. Williams, Titanium, second ed., Springer-Verlag, Berlin, 2007.

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[28] L.E. Murr, S.A. Quinones, S.M. Gaytan, M.I. Lopez, A. Rodela, E.Y. Martinez, D.H. Hernandez, E. Martinez, F. Medina, R.B. Wicker, J. Mech. Behav. Biomed. Mater. (2009) 20. [29] S.Q. Wu, Y.J. Lu , Y.L. Gan, T.T. Huang, C.Q. Zhao , J.J. Lin , S. Guo, J.X. Lin, Journal of

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Alloys and Compounds 672 (2016) 643-652. [30] Y. J. Lu, S. Q. Wu , Y.L. Gan, J. L. Li, C. Q. Zhao, D. X. Zhuo, J.X. Lin, Mater. Sci. Eng. C 49 (2015) 517–525. [

31] ISO 10271:2001. Dentistry-Corrosion test methods for metallic materials.

[32] C.N. Cao, Principle of electrochemical corrosion, Chemical Industry Press, Beijing, 2004. [33] QB/T 2591-2003 Antimicrobial plastics-test for antimicrobial activity and efficacy. [34] GB 4789.2-2010 National food safety standard food microbiological examination: aerobic plate count. [35] ISO-10993-5. Biological evaluation of medical devices-Part 5: Tests for in vitro cytotoxicity. Arlington, VA: ANSI/AAMI, 2009.

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[36] ISO 10993-12. Biological evaluation of medical devices-Part 12: Samples preparation and reference materials. Arlington, VA: ANSI/AAMI, 2009. [37] X. Wu, H. Ma, S. Chen, Z. Xu, A. Sui, J. Electrochem. Soc. 146, 1847–1853 (1999). [38] I. Cvijovic -Alagic, Z. Cvijovic, J. Bajat, M. Rakin, Corros Sci 2014;83:245-54. [39] http://en.wikipedia.org/wiki/Copper in health. [40] L. Ren, K. Yang, L. Guo, H.W. Chai, Mater. Sci. Eng. C 32 (2012) 1204–1209.

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[41] http://www.crct.polymtl.ca/FACT/documentation. [42] R. Cunningham, S. Narra, T. Ozturk, J. Beuth, A.D. Rollett, The Minerals, Metals &

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Materials Society JOM Vol. 68, No. 3 2016.

[43] H. Gong, K. Rafi, G. Hengfeng, T. Starr, and B. Stucker, Addit. Manuf. 1, 87 (2014).

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[44] WHO/FAO/IAEA, (1996), Trace Elements in Human Nutrition and Health. World Health

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Organization, Geneva.

[45] S. Karimi, T. Nickchi, A. Alfantazi, Corros Sci 2011; 53: 3262–72. [46] M. Niinomi, Metall Mater Trans A 2002; 33: 477–86.

MA

[47] M. Takahashi, M. Kikuchi, Y. Takada, O. Okuno, T. Okabe, Dent. Mater. J. 23 (2004) 109. [48] W.C. Rodrigues, L.R. Broilo, L. Schaeffer, G. Knörnschild, F.R.M. Espinoza, Powder Technol.

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206 (2011) 233–238.

[49] Sterritt, RM; Lester, JN (1980). "Interactions of heavy metals with bacteria". The Science of

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the total environment 14 (1): 5–17.

[50] S. Wang, C.G. Yang, L. Ren, M.G. Shen, K. Yang, Mater. Lett. 129 (2014) 88–90. [51] R. Uauy, M. Olivares, M. Gonzalez, Am J Clin Nutr 1998;67:952S–959S.

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[52] E.L. Zhang, L.L. Zheng, J. Liu, B. Bai, C.Liu, Mater. Sci. Eng. C 46 (2015) 148-157.