Materials Science and Engineering C 35 (2014) 392–400
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Effect of Cu content on the antibacterial activity of titanium–copper sintered alloys Jie Liu a,c, Fangbing Li a, Cong Liu a, Hongying Wang a, Baorui Ren a, Ke Yang d, Erlin Zhang a,b,⁎ a
Jiamusi University, Jiamusi 154007, PR China Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, Northeastern University, Shenyang 110819, PR China Dept. Prosthodontics, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, PR China d Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, PR China b c
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 15 October 2013 Accepted 16 November 2013 Available online 1 December 2013 Keywords: Ti–Cu alloy Antibacterial activity Cu-rich phase Galvanic corrosion Antibacterial alloy
a b s t r a c t The phase constitution and the microstructure Ti–x Cu (x = 2, 5, 10 and 25 wt.%) sintered alloys were investigated by XRD and SEM and the antibacterial activity was assessed in order to investigate the effect of the Cu content on the antibacterial activity. The results have shown that Ti2Cu was synthesized as a main secondary phase in all Ti–Cu alloys while Cu-rich phase was formed in the alloys with 5 wt.% or more copper. Antibacterial tests have showed that the Cu content influences the antibacterial rate seriously and only the alloys with 5 wt.% or high Cu have a strong and stable antibacterial rate, which indicates that the Cu content in Ti–Cu alloys must be at least 5 wt.% to obtain strong and stable antibacterial property. The Cu content also influenced the Cu ion release behavior. High Cu ion release concentration and high Cu ion release rate were observed for Ti–Cu alloys with high Cu content. It was concluded that the Cu content affects the Cu existence and the Cu ion release behavior, which in turn influences the antibacterial property. It was thought that the Cu-rich phase should play an important role in the strong antibacterial activity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Titanium and its alloys are widely used in the orthopedic and prosthodontic field because of their excellent biocompatibilities, mechanical characteristics, corrosion resistances and processibility. However, as a kind of bioinert materials, commercial available pure titanium and titanium alloys do not have bactericidal capability shortly after the implantation, the dental plaques can be identified around the implanted dentures [1,2]. The bacterial infection might lead to implant loosening even implantation failure [3]. Common causes of implant-associated infections are Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) [4,5]. Therefore, strict antiseptic operative procedures are adopted to prevent from the clinical infection. From the point view of implant materials, implants with antibacterial activity have been investigated in order to reduce the infection rate, such as antibacterial surface coated titanium implants and antibacterial stainless steel implants. Surface modification has been proven to be an effective way to provide implants with antibacterial activity in the past decades. During these studies, Ag [6–9], Cu [10–12] and Zn [13] were widely used as antibacterial agents. By the addition of Cu element followed by proper heat treatment, antibacterial stainless steel was produced with excellent antibacterial property [14–17]. In our previous ⁎ Corresponding author at: P.O. Box 350, Northeastern University, No 3-11 WenHua Road, Shenyang 110819, PR China. E-mail address: [email protected] (E. Zhang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.028
study [18], it has been proven that Ti–10 wt.% Cu sintered alloy exhibited strong antibacterial property against Escherichia coli and S. aureus with an antibacterial rate of 99.9%. Shirai [19] also reported that Ti–(1 wt.% and 5 wt.%) Cu alloys showed antibacterial property with an antibacterial rate of 30%. In the studies of the antibacterial activity, it has been confirmed that the antibacterial activity of a Cu-surface modified cp-Ti [10] and Cu-containing steel [20] is strongly dependent on the Cu content. Recently, copper element was selected as an alloying element to produce a low melting point cast alloy for dental application [21–24]. It was reported that the copper content in the titanium alloys influenced significantly the mechanical properties, such as elongation, modulus and hardness. In addition, titanium alloy with small amount of copper is reported to have adequate biocompatibility [25] and corrosion resistance for dental use [26,27]. However, excess copper intake causes stomach upset, nausea, and diarrhea and can lead to tissue injury and disease [28]. From above analysis, Ti–Cu alloy is considered as a candidate dental material and shows antibacterial activity. The Cu content in Ti–Cu not only affects the mechanical properties of the alloy, but also determines the final antibacterial activity and the biocompatibility. It is always desired to prepare Ti–Cu alloy with good antibacterial property and good mechanical property. Therefore, in this paper, the effect of the Cu content on the antibacterial property and mechanical property as well as the Cu ion release behavior has been investigated to understand the relationship between the chemical composition, the microstructure and
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phase constitute, the mechanical property and the antibacterial activity and to reveal the controlling antibacterial mechanism. 2. Materials and methods 2.1. Preparation of Ti–Cu alloy High purity titanium powder (99.99%) and 2 wt.%, 5 wt.%, 10 wt.% and 25 wt.% high purity copper powder (99.99%) were ball milled for 3–6 h, then hot pressure sintered under vacuum condition under 5– 35 MPa pressure at 850–1080 °C for 30–60 min. The sample (named Ti–Cu sample) with 25 mm diameter and 1 mm thickness were directly sliced from the sintered Ti–Cu sample for further experiments. Commercial pure titanium sample with similar dimension was used as control sample. Before testing, all samples were ground with SiC paper up to 1000 grits and polished with 1 μm polishing liquid. 2.2. Phase identification and microstructure Phase identification was carried out on D/MAX-RB Rigaku X-ray diffraction (XRD) with a scan step of 0.04. Microstructure was observed on a JSM-6360LV scanning electronic microscope (SEM) with energy dispersive X-ray spectroscopy (EDS). 2.3. Hardness and compressive test Hardness was conducted on a HR-150A Rockwell hardness meter (Huayin, China). The test load was 1470 N and the duration time was 30 s. Five different fields were selected randomly for hardness measurement and the result was a mean value with standard deviation. Compressive strength was conducted with reference to ASTM E9891(2000). Samples with a diameter of 10 mm and a height of 15 mm were cut from the sintered sample. The testing was carried out on a Suns Testing System with a crosshead speed of 0.5 mm/min. At least five samples were tested for each condition. cp-Ti samples with the same dimension were used for comparison.
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2.6. Antibacterial properties Nutrient broth (NB) was prepared by dissolving 10.0 g peptone, 5.0 g beef extract, 5.0 g NaCl and 15.0 g agar in 1000 mL distilled water and the ph value was adjusted to 7.2 to 7.4. NB were then sterilized by autoclaving at 121 °C for 20 min. S. aureus, strain ATCC 6538 and gram negative E. coli, strain ATCC 25922 were used in this study. S. aureus and E. coli were cultivated at 37 °C in the nutrient broth to a concentration of 106 cfu/mL, and then were diluted 10-fold by PBS solution to a concentration of 104 cfu/mL. Plate counting method was conducted with reference to Nation Standard of China GB/T 2591(JIS Z 2801-2000, ASTM G21-96, NEQ) [30]. The Ti–Cu sample and the control sample were placed in Petri dishes separately while nutrient agar was spread onto a Petri dish as a negative blank sample. Then, 0.4 mL of the bacterial suspension was dripped onto the samples. After this, the dishes were covered with a relatively large Petri dish and 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 3.6 mL sterilized physiological saline solution washing. The samples were carefully washed in order to make sure that no bacterium was left on the sample. 0.02 mL solution was selected from the above washing solution and then inoculated onto nutrient agar plates and incubated at 37 °C for 4 h under a humidity of 90%. The active bacteria were counted in accordance with National Standard of China (GB/T 4789.2-2010) [31]. Three samples were assessed for each type of samples. The antibacterial rate R was calculated by the following formula: R ¼ Ncontrol −Nsample =Ncontrol 100%
ð2Þ
where, Ncontrol and Nsample are the average numbers of the bacterial colony on the control sample and the Ti–Cu sample, respectively. 3. Results 3.1. Microstructure
2.4. Electrochemical test Ti–Cu and cp-Ti specimens for the electrochemical test were put into a sample holder with only one side of 10 mm in diameter exposed. Electrochemical test was carried out at 37 ± 1 °C in a beaker containing 500 ml 0.9% NaCl solution on a Versa STAT V3-400 automatic laboratory corrosion measurement system (Princeton Applied Research, USA) using a standard three-electrode configuration with a saturated calomel as a reference, a platinum electrode as a counter and the sample as a working electrode. According to ISO 10271:2001 Standard, the opencircuit potential vs. time curve was recorded for up to 1 h to determine the open-circuit potential (EOCP). The potentiodynamic scan was started 5 min after finishing the open-circuit potential measurement at a scanning rate of 0.5 mV s−1. The corrosion rate (V) was calculated by [29]: V ¼ MI=nF
Fig. 1 shows the XRD patterns of Ti–Cu alloys with different Cu contents. At 2 wt.% Cu, as shown in Fig. 1a, the diffraction peaks of Ti2Cu phase were observed beside the diffraction peaks of titanium matrix. With the increasing Cu content, as shown in Fig. 1b to d, the diffraction intensity of Ti2Cu phase increases, indicating that more Ti2Cu phases were synthesized. Fig. 2 shows the microstructure of Ti–Cu alloys with different Cu contents. At 2 wt.% Cu, as shown in Fig. 2a, gray phases with flake
ð1Þ
where M is the molar mass of titanium (g mol−1), I is the average corrosion current density measured in the electrochemical tests (A cm−2), F is Faraday constant (96,485 C mol−1) and n is the valence of titanium. 2.5. Cu ion release To examine Cu ion release, Ti–Cu sample was immersed in 3.7 mL 0.9% NaCl solution at 37 °C for 24 h to 120 h with a surface area-tovolume ratio of 2.86 with reference of ISO Standard 10993-12:2002 and ISO Standard 10993-15:2000. The Cu ion concentration in the solution was analyzed by an inductively coupled plasma spectrometry (Perkin Elmer, Optima 5300DV) with an accuracy of 0.005 mg/L.
Fig. 1. XRD patterns of Ti–Cu alloys with different Cu contents. a) 2 wt.% Cu, b) 5 wt.% Cu c) 10 wt.% Cu, and d) 25 wt.% Cu.
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Fig. 2. SEM microstructure of Ti–Cu alloys. a) Ti–2.0wt.% Cu, b) Ti–5.0wt.% Cu, c) Ti–10.0wt.% Cu, and d) Ti–25.0wt.% Cu.
shape distribute mainly along the grain boundary. EDS analysis results (not shown here) on the gray phases indicate these phases constitute of 69.4 at.% Ti and 30.6 at.% Cu, corresponding to Ti2Cu phase in combination with XRD pattern (Fig. 1a). At 5 wt.% Cu, many gray phases with laminate structure can be observed on the matrix. As an example, Fig. 3a and b shows a high magnification microstructure and EDS result of Ti– 5 wt.% Cu alloy. Some phases with high Cu content were detected beside Ti2Cu phases, e.g. 40.08 at.% Cu at the cross point A. At 10 wt.% Cu, more gray phases with laminate structure are found on the matrix. High magnification SEM microstructure and EDS result (not shown here) indicate that some phases with high Cu content were also detected. At 25 wt.% Cu, large amount of gray phases with block shape rather than laminate structure can be found on the titanium matrix. EDS analysis results on these phases also show high Cu content, e.g. 44.6 at.% Cu as shown in Fig. 3c and d. 3.2. Hardness and compressive strength Fig. 4 shows the change of the hardness of Ti–Cu alloys with Cu content. The hardness increases with the increasing Cu content and reaches a high value of 55HRC at 2 wt.% Cu. Further increase in Cu content does not change the hardness significantly. However, a slight drop can be found in the hardness at 10 wt.% Cu. With the consideration of deviation, the hardness of Ti–Cu alloys with 5 wt.%, 10 wt.% and 25 wt.% Cu is at a nearly the same level. This drop is considered to be due to the measurement deviation. Fig. 5 illustrates typical stress–strain curves of pure Ti and Ti–Cu alloys in the compressive testing. cp-Ti displays low yield compressive strength and very good ductility. Even after 55% strain, no crack was observed on the samples. Ti–Cu alloys show significant high yield compressive strength and compressive strength, but low ductility, which demonstrates that the addition of Cu considerably enhances the strength of titanium matrix, but on the other hand deteriorates the ductility significantly. Ti– 2 wt.% Cu shows the best ductility among Ti–Cu sintered alloys, about 25–28%. But no difference is found in the ductility among Ti–Cu sintered alloys with 5–25 wt.% Cu and the ductility is about 15%.
Fig. 6 presents the change of compressive strength as well as yield compressive strength of Ti and Ti–Cu alloys with different Cu contents. The yield compressive strength of cp-Ti alloy is about 439 MPa and the compressive strength is not available because the sample was not broken. For Ti–Cu samples, the yield strength increases rapidly with the increasing Cu content and arrives at the maximum value at 5 wt.% Cu and then gradually decreases with further increase in Cu content. Meanwhile, the compressive strength of Ti–Cu decreases with the increasing Cu content. 3.3. Corrosion properties and Cu ion release Fig. 7 shows typical OCP curves and Tafel curves of Ti–Cu alloys with different Cu contents. Table 1 lists the electrochemical data obtained from the OCP and Tafel curves. Eocp and Ecorr of Ti−Cu alloys are both nobler while the corrosion current densities are lower than the values of cp-Ti. The calculated corrosion rate also indicates that Ti–Cu alloys exhibit lower corrosion rate than cp-Ti, displaying that the addition of Cu element could slightly improve the biocorrosion resistance of Ti. Although no difference can be found in Eocp and Ecorr of Ti–Cu alloys, the corrosion current density decreases with the increasing Cu content. Fig. 8 shows the cumulative Cu ion concentration and the Cu ion release rate from Ti–Cu alloys during up to 120 h immersion in 0.9% NaCl solution. The cumulative Cu ion concentration increases with the increasing Cu content and with the extension of immersion duration. Fast Cu ion release was observed at the first 24 h immersion, as shown in Fig. 8b, and then the release rate decreases with further immersion up to 120 h. Ti–Cu alloys with high Cu content exhibits high Cu ion release rate and high Cu ion concentration, especially at the early immersion stage. 3.4. Antibacterial activity Fig. 9 shows the E. coli colonies incubated for 24 h on the negative sample, cp-Ti (the control sample) and Ti–Cu alloys with different Cu contents. In Fig. 9a and b, large amount of bacteria are observed on
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Fig. 3. High magnification microstructure and EDS analysis results of Ti–5 wt.% Cu and Ti–25 wt.% Cu alloys. a) High magnification microstructure of Ti–5wt.% Cu, showing the laminate structure, b) EDS analysis result at the cross point A in panel a, c) high magnification microstructure of Ti–25wt.% Cu alloy, showing the blocky phases, and d) EDS analysis result at the cross point in panel c.
the negative sample and cp-Ti sample, corresponding to the fact that cpTi does not have antibacterial activity. Lots of bacteria are also found on Ti–2.0 wt.% Cu sample, as shown in Fig. 9c, displaying that this alloy has a low antibacterial activity. On Ti–5 wt.% Cu sample, only a few bacteria are found, as shown in Fig. 9d while nearly no bacteria is found on Ti– 10 wt.% Cu and Ti–25 wt.% Cu samples, as shown in Fig. 9e and f, indicating that Ti–5 wt.% Cu, Ti–10 wt.% Cu and Ti–25 wt.% Cu samples have strong antibacterial activity. Similar results are found in the experiment against S. aureus: lots of bacteria on the negative sample and the control sample as well as Ti–2.0 wt.% Cu sample, but only a few bacteria or no bacteria on other three samples, as shown in Fig. 10. Fig. 11 shows the change of the calculated antibacterial rate of Ti–Cu alloys with different Cu contents. The average antibacterial rates of Ti–
Fig. 4. Change of hardness with the Cu content in the Ti–Cu alloys.
2.0 wt.% Cu against E. coli and S. aureus are 57% and 79%, respectively, less than 90%, indicating the alloy does not have antibacterial activity according to Standard (GB/T 4789.2-2010) [31]. It has to be pointed out that the standard deviations are very high, 48% and 28%, respectively, displaying that the antibacterial activity of this alloy is not stable. When the Cu content rises to 5 wt.%, the antibacterial rate sharply jumps to a very high value, as high as 99.2% against E. coli and 99.0% against S. aureus, respectively, meaning that this alloy has antibacterial property. When the Cu content increases to 10 wt.% and 25 wt.%, the antibacterial rates are larger than 99.99%, meaning that these alloys have strong antibacterial activity. It can be deduced from the above results that the Cu content has a significant influence on the antibacterial property and the Cu content in Ti–Cu alloy has to be at least 5 wt.% in order to get a stable antibacterial activity.
Fig. 5. Typical stress–strain curves of pure Ti and Ti–Cu alloys in compressive strength testing.
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J. Liu et al. / Materials Science and Engineering C 35 (2014) 392–400 Table 1 Electrochemical data of cp-Ti and Ti–Cu alloys with different Cu contents.
Fig. 6. Change of 0.2% yield compressive strength and compressive strength with Cu content in the Ti–Cu alloys.
4. Discussion In the binary phase diagram of Ti–Cu, only Ti2Cu phase will be synthesized as a secondary phase in Ti–Cu alloy with less than 40 wt.% Cu under an equilibrium solidification condition [32]. In this study, titanium and copper powders were sintered at a temperature less than 1080 °C. Under this temperature, titanium powder and copper powder both are at solid state, which displays that only a solid reaction happened between Ti powder and Cu powder during the sintering process.
Fig. 7. Typical OCP (a) and Tafel curves (b) of Ti–Cu alloys with different Cu contents.
Samples
Eocp at 1 h (V)
Ecorr (V)
icorr (μ A/cm−2)
Corrosion rate (mg/(cm2 yr))
cp-Ti Ti–2.0 wt.% Cu Ti–5.0 wt.% Cu Ti–10.0 wt.% Cu Ti–25.0 wt.% Cu
−0.292 −0.160 −0.281 −0.186 −0.217
−0.398 −0.198 −0.281 −0.206 −0.238
3.831 1.115 0.879 0.552 0.139
17.4 8.7 6.9 4.3 1.1
XRD, SEM and EDS results indicate that only Ti2Cu phase was synthesized in Ti–2 wt.% Cu. However, in other Ti–Cu alloys with 5 wt.% Cu, 10 wt.% Cu and 25 wt.% Cu, other intermetallic phase with high Cu content (named Cu-rich phase), e.g. 40–44 at.% Cu, was also detected, displaying that solid state reaction in these alloys was not complete due to the low sintering temperature and short sintering duration. The Cu-rich phase is not an equilibrium phase and does not have a stoichiometric composition, therefore, the Cu content in this phase changes depending on the sintering temperature, the sintering duration as well as the Cu content in the whole alloy and the size of Cu powder. The mechanical property of a biomedical material is of crucial importance in the clinical application. Previous study [33] indicated that Ti–Cu cast alloys containing 10% or more copper has minimal ductility. On the contrary, heat-treated Ti–13% Cu displayed excellent ductility of 12% [34]. The formation of Ti2Cu phase and Cu-rich phase in Ti–Cu sintered alloys enhances the strength but reduces the ductility seriously. It is very necessary to improve the ductility of Ti–Cu alloy by some ways in the next step. The mechanical properties of a titanium alloy are closely
Fig. 8. Cumulative Cu ion concentration (a) and Cu ion release rate from Ti–Cu alloys (b).
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Fig. 9. E. coli bacterial colonies after incubation for 24 h on different samples. a) Negative sample, b) control sample, c) Ti–2 wt.% Cu, d) Ti–5.0 wt.% Cu, e) Ti–10.0 wt.%, and f) Ti–25wt.% Cu.
related to the chemical composition and the microstructure, including the microstructure of the matrix, the formation, the size and the distribution of the secondary phase. Ti–Cu alloys were prepared by a powder metallurgy in this paper. Therefore, the preparation parameters, including the ball milling process and the sintering process determine the microstructure of the final alloy, thus in turn change the mechanical property. In addition, hot forming processing, such as extrusion, rolling and forging can also refine the microstructure and improve the mechanical property.
Good corrosion resistance is one of the most important reasons for titanium and titanium alloys to be applied as orthopedic and dental implant. Electrochemical testing demonstrates that Cu–Ti alloys are of slightly better anticorrosion property than cp-Ti. Cu ion release measurements clearly demonstrate that the Cu ion concentration linearly increases with the extension of immersion for all test alloys. However, the Cu ion release rate linearly decreases with the extension of immersion, as shown in Fig. 8b. The metal ion is released by the reaction between Ti–Cu alloy and the immersion solution, which happens on the
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Fig. 10. S. aureus bacterial colonies after incubation for 24 h on different samples. a) Negative sample, b) control sample. c) Ti–2 wt.% Cu, d) Ti–5.0 wt.% Cu, e) Ti–10.0 wt.%, and f) Ti–25wt.% Cu.
surface of Ti–Cu alloy. Therefore, only the Cu-containing compounds on the surface will react with the solution, which results in the metal ion release. With the increasing immersion duration, a passive layer might be formed on the surface due to above reaction, which reduces the metal ion release rate as observed in Fig. 8b. Shirai et al. [19] reported that Ti–(1 wt.%, 5 wt.%) Cu alloys had antimicrobial activity, but no difference was found in the antibacterial rates of pure titanium and Ti–1.0 wt.% Cu alloy and the antibacterial rate of Ti–5wt.% Cu alloy against E. coli was only about 30% (calculated from the data in Fig. 4 in reference [19]). In this paper, Ti–2 wt.% Cu alloy
shows an unstable antibacterial activity with an antibacterial rate of 57–80%. Alloys with 5 wt.% or more copper show not only high antibacterial rate but also stable antibacterial activity. The above results indicate that the Cu content influences the antibacterial activity and the crucial Cu content should be at least 5 wt.% in order to get a stable and high antibacterial property. Although the mechanism of inhibitory action of Cu containing materials is only partially known, it is believed that Cu ion released from Cu containing material plays an important role. Cu ion might alter the structure of proteins [35], or disrupt enzyme structure and functions
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between the chemical composition, microstructure, mechanical property and antibacterial activity suggests that it should be possible to get comprehensive property by optimizing the chemical composition and microstructure. Also the fatigue property of Ti–Cu under biological environment needs to be assessed for clinical application. 5. Conclusion Ti–Cu alloys exhibited antibacterial property against E. coli and S. aureus. The Cu content affects the existence form of Cu in the Ti–Cu, the Cu ion release, and in turn influences the antibacterial activity seriously. The crucial Cu content in Ti–Cu alloys should be at least 5 wt.% in order to obtain stable and strong antibacterial activity against E. coli and S. aureus bacteria. The bacterial mechanism was thought to be related to the Cu ion release due to the galvanic corrosion between Cu-rich phase and titanium matrix. Fig. 11. Antibacterial rate of Ti–Cu alloys against E. coli and S. aureus with change in Cu content.
by binding to sulfur- or carboxylate-containing groups [36], or facilitate deleterious activity in superoxide radicals and cause “multiple hit damage” at target sites [37]. Our results also indicate that only Ti–Cu alloys with more than 5 wt.% Cu exhibits strong antibacterial activity, proving again that the antibacterial activity is really dependent on the Cu content. Fig. 12 illustrates the relationship between the antibacterial rate and the Cu ion concentration in the immersion solution. When the Cu ion concentration in the immersion solution is more than 0.036 mg/L, the antibacterial rate is larger than 99%, which is the recommended value for a strong antibacterial material [30]. Therefore, it is possible to predict the antibacterial property of Ti–Cu alloys by measuring the Cu ion release concentration in the immersion solution. In Ti–2.0 wt.% Cu, only Ti2Cu phase was formed and the Cu ion concentration kept at a low level, 0.0176 mg/L after 24 h immersion while the antibacterial rate is very low. In Ti–Cu alloys with 5 wt.% or more copper, Cu-rich phase besides Ti2Cu phase was formed and the Cu ion concentration kept at a relatively high level even after 24 h immersion, which responds to a high antibacterial rate. From the above results, it can be deduced that the Cu-rich phase plays a very important role in the antibacterial property of Ti–Cu alloys. The above results demonstrate that the Cu content in Ti–Cu sintered alloys seriously influences the existence form of Cu and the Cu ion release behavior, and in turn affects the mechanical property and the antibacterial property. In order to get strong and stable antibacterial property, the Cu content has to be at least 5 wt.% in Ti–Cu sintered alloy. However, the low ductility of Ti–Cu alloys will be a big problem for the clinical application. Above understanding on the relationship
Fig. 12. Relationship between the antibacterial rate and the Cu ion concentration in immersion solution.
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[28] http://en.wikipedia.org/wiki/Copper_in_health. [29] C.N. Cao, Principle of Electrochemical Corrosion, Chemical Industry Press, Beijing, 2004. [30] QB/T 2591-2003 Antimicrobial plastics—test for antimicrobial activity and efficacy. [31] GB 4789.2-2010 National food safety standard food microbiological examination: aerobic plate count. [32] T.B. Massalski, Binary Alloy Phase Diagram, ASM International, Materials Park, Ohio, 1990. [33] F.C. Holden, A.A. Watts, H.R. Ogden, R.I. Jaffee, Heat treatment and mechanical properties of Ti–Cu alloys, Trans. AIME 7 (1955) 117–125. [34] D.R. Schuyler, J.A. Petrusha, Investment casting of low-melting titanium alloy, in: R.C. Krutenat (Ed.), Vacuum Metallurgy, Science Press, Princeton, 1977, pp. 475–503. [35] R.B. Thurman, C.P. Gerba, The molecular mechanisms of copper and silver ion disinfection of bacteria and viruses, CRC Crit. Rev. Environ. Control. 18 (1989) 295–315. [36] R. Sterritt, J. Lester, Interactions of heavy metals with bacteria, Sci. Total Environ. 14 (1980) 5–17. [37] A. Samuni, M. Chevion, G. Czapski, Roles of copper and superoxide anion radicals in the radiation-induced inactivation of T7 bacteriophage, Radiat. Res. 99 (1984) 562–572.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Effect of Cu content on the antibacterial activity of titanium–copper sintered alloys Jie Liu a,c, Fangbing Li a, Cong Liu a, Hongying Wang a, Baorui Ren a, Ke Yang d, Erlin Zhang a,b,⁎ a
Jiamusi University, Jiamusi 154007, PR China Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, Northeastern University, Shenyang 110819, PR China Dept. Prosthodontics, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, PR China d Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, PR China b c
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 15 October 2013 Accepted 16 November 2013 Available online 1 December 2013 Keywords: Ti–Cu alloy Antibacterial activity Cu-rich phase Galvanic corrosion Antibacterial alloy
a b s t r a c t The phase constitution and the microstructure Ti–x Cu (x = 2, 5, 10 and 25 wt.%) sintered alloys were investigated by XRD and SEM and the antibacterial activity was assessed in order to investigate the effect of the Cu content on the antibacterial activity. The results have shown that Ti2Cu was synthesized as a main secondary phase in all Ti–Cu alloys while Cu-rich phase was formed in the alloys with 5 wt.% or more copper. Antibacterial tests have showed that the Cu content influences the antibacterial rate seriously and only the alloys with 5 wt.% or high Cu have a strong and stable antibacterial rate, which indicates that the Cu content in Ti–Cu alloys must be at least 5 wt.% to obtain strong and stable antibacterial property. The Cu content also influenced the Cu ion release behavior. High Cu ion release concentration and high Cu ion release rate were observed for Ti–Cu alloys with high Cu content. It was concluded that the Cu content affects the Cu existence and the Cu ion release behavior, which in turn influences the antibacterial property. It was thought that the Cu-rich phase should play an important role in the strong antibacterial activity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Titanium and its alloys are widely used in the orthopedic and prosthodontic field because of their excellent biocompatibilities, mechanical characteristics, corrosion resistances and processibility. However, as a kind of bioinert materials, commercial available pure titanium and titanium alloys do not have bactericidal capability shortly after the implantation, the dental plaques can be identified around the implanted dentures [1,2]. The bacterial infection might lead to implant loosening even implantation failure [3]. Common causes of implant-associated infections are Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) [4,5]. Therefore, strict antiseptic operative procedures are adopted to prevent from the clinical infection. From the point view of implant materials, implants with antibacterial activity have been investigated in order to reduce the infection rate, such as antibacterial surface coated titanium implants and antibacterial stainless steel implants. Surface modification has been proven to be an effective way to provide implants with antibacterial activity in the past decades. During these studies, Ag [6–9], Cu [10–12] and Zn [13] were widely used as antibacterial agents. By the addition of Cu element followed by proper heat treatment, antibacterial stainless steel was produced with excellent antibacterial property [14–17]. In our previous ⁎ Corresponding author at: P.O. Box 350, Northeastern University, No 3-11 WenHua Road, Shenyang 110819, PR China. E-mail address: [email protected] (E. Zhang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.028
study [18], it has been proven that Ti–10 wt.% Cu sintered alloy exhibited strong antibacterial property against Escherichia coli and S. aureus with an antibacterial rate of 99.9%. Shirai [19] also reported that Ti–(1 wt.% and 5 wt.%) Cu alloys showed antibacterial property with an antibacterial rate of 30%. In the studies of the antibacterial activity, it has been confirmed that the antibacterial activity of a Cu-surface modified cp-Ti [10] and Cu-containing steel [20] is strongly dependent on the Cu content. Recently, copper element was selected as an alloying element to produce a low melting point cast alloy for dental application [21–24]. It was reported that the copper content in the titanium alloys influenced significantly the mechanical properties, such as elongation, modulus and hardness. In addition, titanium alloy with small amount of copper is reported to have adequate biocompatibility [25] and corrosion resistance for dental use [26,27]. However, excess copper intake causes stomach upset, nausea, and diarrhea and can lead to tissue injury and disease [28]. From above analysis, Ti–Cu alloy is considered as a candidate dental material and shows antibacterial activity. The Cu content in Ti–Cu not only affects the mechanical properties of the alloy, but also determines the final antibacterial activity and the biocompatibility. It is always desired to prepare Ti–Cu alloy with good antibacterial property and good mechanical property. Therefore, in this paper, the effect of the Cu content on the antibacterial property and mechanical property as well as the Cu ion release behavior has been investigated to understand the relationship between the chemical composition, the microstructure and
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phase constitute, the mechanical property and the antibacterial activity and to reveal the controlling antibacterial mechanism. 2. Materials and methods 2.1. Preparation of Ti–Cu alloy High purity titanium powder (99.99%) and 2 wt.%, 5 wt.%, 10 wt.% and 25 wt.% high purity copper powder (99.99%) were ball milled for 3–6 h, then hot pressure sintered under vacuum condition under 5– 35 MPa pressure at 850–1080 °C for 30–60 min. The sample (named Ti–Cu sample) with 25 mm diameter and 1 mm thickness were directly sliced from the sintered Ti–Cu sample for further experiments. Commercial pure titanium sample with similar dimension was used as control sample. Before testing, all samples were ground with SiC paper up to 1000 grits and polished with 1 μm polishing liquid. 2.2. Phase identification and microstructure Phase identification was carried out on D/MAX-RB Rigaku X-ray diffraction (XRD) with a scan step of 0.04. Microstructure was observed on a JSM-6360LV scanning electronic microscope (SEM) with energy dispersive X-ray spectroscopy (EDS). 2.3. Hardness and compressive test Hardness was conducted on a HR-150A Rockwell hardness meter (Huayin, China). The test load was 1470 N and the duration time was 30 s. Five different fields were selected randomly for hardness measurement and the result was a mean value with standard deviation. Compressive strength was conducted with reference to ASTM E9891(2000). Samples with a diameter of 10 mm and a height of 15 mm were cut from the sintered sample. The testing was carried out on a Suns Testing System with a crosshead speed of 0.5 mm/min. At least five samples were tested for each condition. cp-Ti samples with the same dimension were used for comparison.
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2.6. Antibacterial properties Nutrient broth (NB) was prepared by dissolving 10.0 g peptone, 5.0 g beef extract, 5.0 g NaCl and 15.0 g agar in 1000 mL distilled water and the ph value was adjusted to 7.2 to 7.4. NB were then sterilized by autoclaving at 121 °C for 20 min. S. aureus, strain ATCC 6538 and gram negative E. coli, strain ATCC 25922 were used in this study. S. aureus and E. coli were cultivated at 37 °C in the nutrient broth to a concentration of 106 cfu/mL, and then were diluted 10-fold by PBS solution to a concentration of 104 cfu/mL. Plate counting method was conducted with reference to Nation Standard of China GB/T 2591(JIS Z 2801-2000, ASTM G21-96, NEQ) [30]. The Ti–Cu sample and the control sample were placed in Petri dishes separately while nutrient agar was spread onto a Petri dish as a negative blank sample. Then, 0.4 mL of the bacterial suspension was dripped onto the samples. After this, the dishes were covered with a relatively large Petri dish and 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 3.6 mL sterilized physiological saline solution washing. The samples were carefully washed in order to make sure that no bacterium was left on the sample. 0.02 mL solution was selected from the above washing solution and then inoculated onto nutrient agar plates and incubated at 37 °C for 4 h under a humidity of 90%. The active bacteria were counted in accordance with National Standard of China (GB/T 4789.2-2010) [31]. Three samples were assessed for each type of samples. The antibacterial rate R was calculated by the following formula: R ¼ Ncontrol −Nsample =Ncontrol 100%
ð2Þ
where, Ncontrol and Nsample are the average numbers of the bacterial colony on the control sample and the Ti–Cu sample, respectively. 3. Results 3.1. Microstructure
2.4. Electrochemical test Ti–Cu and cp-Ti specimens for the electrochemical test were put into a sample holder with only one side of 10 mm in diameter exposed. Electrochemical test was carried out at 37 ± 1 °C in a beaker containing 500 ml 0.9% NaCl solution on a Versa STAT V3-400 automatic laboratory corrosion measurement system (Princeton Applied Research, USA) using a standard three-electrode configuration with a saturated calomel as a reference, a platinum electrode as a counter and the sample as a working electrode. According to ISO 10271:2001 Standard, the opencircuit potential vs. time curve was recorded for up to 1 h to determine the open-circuit potential (EOCP). The potentiodynamic scan was started 5 min after finishing the open-circuit potential measurement at a scanning rate of 0.5 mV s−1. The corrosion rate (V) was calculated by [29]: V ¼ MI=nF
Fig. 1 shows the XRD patterns of Ti–Cu alloys with different Cu contents. At 2 wt.% Cu, as shown in Fig. 1a, the diffraction peaks of Ti2Cu phase were observed beside the diffraction peaks of titanium matrix. With the increasing Cu content, as shown in Fig. 1b to d, the diffraction intensity of Ti2Cu phase increases, indicating that more Ti2Cu phases were synthesized. Fig. 2 shows the microstructure of Ti–Cu alloys with different Cu contents. At 2 wt.% Cu, as shown in Fig. 2a, gray phases with flake
ð1Þ
where M is the molar mass of titanium (g mol−1), I is the average corrosion current density measured in the electrochemical tests (A cm−2), F is Faraday constant (96,485 C mol−1) and n is the valence of titanium. 2.5. Cu ion release To examine Cu ion release, Ti–Cu sample was immersed in 3.7 mL 0.9% NaCl solution at 37 °C for 24 h to 120 h with a surface area-tovolume ratio of 2.86 with reference of ISO Standard 10993-12:2002 and ISO Standard 10993-15:2000. The Cu ion concentration in the solution was analyzed by an inductively coupled plasma spectrometry (Perkin Elmer, Optima 5300DV) with an accuracy of 0.005 mg/L.
Fig. 1. XRD patterns of Ti–Cu alloys with different Cu contents. a) 2 wt.% Cu, b) 5 wt.% Cu c) 10 wt.% Cu, and d) 25 wt.% Cu.
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Fig. 2. SEM microstructure of Ti–Cu alloys. a) Ti–2.0wt.% Cu, b) Ti–5.0wt.% Cu, c) Ti–10.0wt.% Cu, and d) Ti–25.0wt.% Cu.
shape distribute mainly along the grain boundary. EDS analysis results (not shown here) on the gray phases indicate these phases constitute of 69.4 at.% Ti and 30.6 at.% Cu, corresponding to Ti2Cu phase in combination with XRD pattern (Fig. 1a). At 5 wt.% Cu, many gray phases with laminate structure can be observed on the matrix. As an example, Fig. 3a and b shows a high magnification microstructure and EDS result of Ti– 5 wt.% Cu alloy. Some phases with high Cu content were detected beside Ti2Cu phases, e.g. 40.08 at.% Cu at the cross point A. At 10 wt.% Cu, more gray phases with laminate structure are found on the matrix. High magnification SEM microstructure and EDS result (not shown here) indicate that some phases with high Cu content were also detected. At 25 wt.% Cu, large amount of gray phases with block shape rather than laminate structure can be found on the titanium matrix. EDS analysis results on these phases also show high Cu content, e.g. 44.6 at.% Cu as shown in Fig. 3c and d. 3.2. Hardness and compressive strength Fig. 4 shows the change of the hardness of Ti–Cu alloys with Cu content. The hardness increases with the increasing Cu content and reaches a high value of 55HRC at 2 wt.% Cu. Further increase in Cu content does not change the hardness significantly. However, a slight drop can be found in the hardness at 10 wt.% Cu. With the consideration of deviation, the hardness of Ti–Cu alloys with 5 wt.%, 10 wt.% and 25 wt.% Cu is at a nearly the same level. This drop is considered to be due to the measurement deviation. Fig. 5 illustrates typical stress–strain curves of pure Ti and Ti–Cu alloys in the compressive testing. cp-Ti displays low yield compressive strength and very good ductility. Even after 55% strain, no crack was observed on the samples. Ti–Cu alloys show significant high yield compressive strength and compressive strength, but low ductility, which demonstrates that the addition of Cu considerably enhances the strength of titanium matrix, but on the other hand deteriorates the ductility significantly. Ti– 2 wt.% Cu shows the best ductility among Ti–Cu sintered alloys, about 25–28%. But no difference is found in the ductility among Ti–Cu sintered alloys with 5–25 wt.% Cu and the ductility is about 15%.
Fig. 6 presents the change of compressive strength as well as yield compressive strength of Ti and Ti–Cu alloys with different Cu contents. The yield compressive strength of cp-Ti alloy is about 439 MPa and the compressive strength is not available because the sample was not broken. For Ti–Cu samples, the yield strength increases rapidly with the increasing Cu content and arrives at the maximum value at 5 wt.% Cu and then gradually decreases with further increase in Cu content. Meanwhile, the compressive strength of Ti–Cu decreases with the increasing Cu content. 3.3. Corrosion properties and Cu ion release Fig. 7 shows typical OCP curves and Tafel curves of Ti–Cu alloys with different Cu contents. Table 1 lists the electrochemical data obtained from the OCP and Tafel curves. Eocp and Ecorr of Ti−Cu alloys are both nobler while the corrosion current densities are lower than the values of cp-Ti. The calculated corrosion rate also indicates that Ti–Cu alloys exhibit lower corrosion rate than cp-Ti, displaying that the addition of Cu element could slightly improve the biocorrosion resistance of Ti. Although no difference can be found in Eocp and Ecorr of Ti–Cu alloys, the corrosion current density decreases with the increasing Cu content. Fig. 8 shows the cumulative Cu ion concentration and the Cu ion release rate from Ti–Cu alloys during up to 120 h immersion in 0.9% NaCl solution. The cumulative Cu ion concentration increases with the increasing Cu content and with the extension of immersion duration. Fast Cu ion release was observed at the first 24 h immersion, as shown in Fig. 8b, and then the release rate decreases with further immersion up to 120 h. Ti–Cu alloys with high Cu content exhibits high Cu ion release rate and high Cu ion concentration, especially at the early immersion stage. 3.4. Antibacterial activity Fig. 9 shows the E. coli colonies incubated for 24 h on the negative sample, cp-Ti (the control sample) and Ti–Cu alloys with different Cu contents. In Fig. 9a and b, large amount of bacteria are observed on
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Fig. 3. High magnification microstructure and EDS analysis results of Ti–5 wt.% Cu and Ti–25 wt.% Cu alloys. a) High magnification microstructure of Ti–5wt.% Cu, showing the laminate structure, b) EDS analysis result at the cross point A in panel a, c) high magnification microstructure of Ti–25wt.% Cu alloy, showing the blocky phases, and d) EDS analysis result at the cross point in panel c.
the negative sample and cp-Ti sample, corresponding to the fact that cpTi does not have antibacterial activity. Lots of bacteria are also found on Ti–2.0 wt.% Cu sample, as shown in Fig. 9c, displaying that this alloy has a low antibacterial activity. On Ti–5 wt.% Cu sample, only a few bacteria are found, as shown in Fig. 9d while nearly no bacteria is found on Ti– 10 wt.% Cu and Ti–25 wt.% Cu samples, as shown in Fig. 9e and f, indicating that Ti–5 wt.% Cu, Ti–10 wt.% Cu and Ti–25 wt.% Cu samples have strong antibacterial activity. Similar results are found in the experiment against S. aureus: lots of bacteria on the negative sample and the control sample as well as Ti–2.0 wt.% Cu sample, but only a few bacteria or no bacteria on other three samples, as shown in Fig. 10. Fig. 11 shows the change of the calculated antibacterial rate of Ti–Cu alloys with different Cu contents. The average antibacterial rates of Ti–
Fig. 4. Change of hardness with the Cu content in the Ti–Cu alloys.
2.0 wt.% Cu against E. coli and S. aureus are 57% and 79%, respectively, less than 90%, indicating the alloy does not have antibacterial activity according to Standard (GB/T 4789.2-2010) [31]. It has to be pointed out that the standard deviations are very high, 48% and 28%, respectively, displaying that the antibacterial activity of this alloy is not stable. When the Cu content rises to 5 wt.%, the antibacterial rate sharply jumps to a very high value, as high as 99.2% against E. coli and 99.0% against S. aureus, respectively, meaning that this alloy has antibacterial property. When the Cu content increases to 10 wt.% and 25 wt.%, the antibacterial rates are larger than 99.99%, meaning that these alloys have strong antibacterial activity. It can be deduced from the above results that the Cu content has a significant influence on the antibacterial property and the Cu content in Ti–Cu alloy has to be at least 5 wt.% in order to get a stable antibacterial activity.
Fig. 5. Typical stress–strain curves of pure Ti and Ti–Cu alloys in compressive strength testing.
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J. Liu et al. / Materials Science and Engineering C 35 (2014) 392–400 Table 1 Electrochemical data of cp-Ti and Ti–Cu alloys with different Cu contents.
Fig. 6. Change of 0.2% yield compressive strength and compressive strength with Cu content in the Ti–Cu alloys.
4. Discussion In the binary phase diagram of Ti–Cu, only Ti2Cu phase will be synthesized as a secondary phase in Ti–Cu alloy with less than 40 wt.% Cu under an equilibrium solidification condition [32]. In this study, titanium and copper powders were sintered at a temperature less than 1080 °C. Under this temperature, titanium powder and copper powder both are at solid state, which displays that only a solid reaction happened between Ti powder and Cu powder during the sintering process.
Fig. 7. Typical OCP (a) and Tafel curves (b) of Ti–Cu alloys with different Cu contents.
Samples
Eocp at 1 h (V)
Ecorr (V)
icorr (μ A/cm−2)
Corrosion rate (mg/(cm2 yr))
cp-Ti Ti–2.0 wt.% Cu Ti–5.0 wt.% Cu Ti–10.0 wt.% Cu Ti–25.0 wt.% Cu
−0.292 −0.160 −0.281 −0.186 −0.217
−0.398 −0.198 −0.281 −0.206 −0.238
3.831 1.115 0.879 0.552 0.139
17.4 8.7 6.9 4.3 1.1
XRD, SEM and EDS results indicate that only Ti2Cu phase was synthesized in Ti–2 wt.% Cu. However, in other Ti–Cu alloys with 5 wt.% Cu, 10 wt.% Cu and 25 wt.% Cu, other intermetallic phase with high Cu content (named Cu-rich phase), e.g. 40–44 at.% Cu, was also detected, displaying that solid state reaction in these alloys was not complete due to the low sintering temperature and short sintering duration. The Cu-rich phase is not an equilibrium phase and does not have a stoichiometric composition, therefore, the Cu content in this phase changes depending on the sintering temperature, the sintering duration as well as the Cu content in the whole alloy and the size of Cu powder. The mechanical property of a biomedical material is of crucial importance in the clinical application. Previous study [33] indicated that Ti–Cu cast alloys containing 10% or more copper has minimal ductility. On the contrary, heat-treated Ti–13% Cu displayed excellent ductility of 12% [34]. The formation of Ti2Cu phase and Cu-rich phase in Ti–Cu sintered alloys enhances the strength but reduces the ductility seriously. It is very necessary to improve the ductility of Ti–Cu alloy by some ways in the next step. The mechanical properties of a titanium alloy are closely
Fig. 8. Cumulative Cu ion concentration (a) and Cu ion release rate from Ti–Cu alloys (b).
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Fig. 9. E. coli bacterial colonies after incubation for 24 h on different samples. a) Negative sample, b) control sample, c) Ti–2 wt.% Cu, d) Ti–5.0 wt.% Cu, e) Ti–10.0 wt.%, and f) Ti–25wt.% Cu.
related to the chemical composition and the microstructure, including the microstructure of the matrix, the formation, the size and the distribution of the secondary phase. Ti–Cu alloys were prepared by a powder metallurgy in this paper. Therefore, the preparation parameters, including the ball milling process and the sintering process determine the microstructure of the final alloy, thus in turn change the mechanical property. In addition, hot forming processing, such as extrusion, rolling and forging can also refine the microstructure and improve the mechanical property.
Good corrosion resistance is one of the most important reasons for titanium and titanium alloys to be applied as orthopedic and dental implant. Electrochemical testing demonstrates that Cu–Ti alloys are of slightly better anticorrosion property than cp-Ti. Cu ion release measurements clearly demonstrate that the Cu ion concentration linearly increases with the extension of immersion for all test alloys. However, the Cu ion release rate linearly decreases with the extension of immersion, as shown in Fig. 8b. The metal ion is released by the reaction between Ti–Cu alloy and the immersion solution, which happens on the
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Fig. 10. S. aureus bacterial colonies after incubation for 24 h on different samples. a) Negative sample, b) control sample. c) Ti–2 wt.% Cu, d) Ti–5.0 wt.% Cu, e) Ti–10.0 wt.%, and f) Ti–25wt.% Cu.
surface of Ti–Cu alloy. Therefore, only the Cu-containing compounds on the surface will react with the solution, which results in the metal ion release. With the increasing immersion duration, a passive layer might be formed on the surface due to above reaction, which reduces the metal ion release rate as observed in Fig. 8b. Shirai et al. [19] reported that Ti–(1 wt.%, 5 wt.%) Cu alloys had antimicrobial activity, but no difference was found in the antibacterial rates of pure titanium and Ti–1.0 wt.% Cu alloy and the antibacterial rate of Ti–5wt.% Cu alloy against E. coli was only about 30% (calculated from the data in Fig. 4 in reference [19]). In this paper, Ti–2 wt.% Cu alloy
shows an unstable antibacterial activity with an antibacterial rate of 57–80%. Alloys with 5 wt.% or more copper show not only high antibacterial rate but also stable antibacterial activity. The above results indicate that the Cu content influences the antibacterial activity and the crucial Cu content should be at least 5 wt.% in order to get a stable and high antibacterial property. Although the mechanism of inhibitory action of Cu containing materials is only partially known, it is believed that Cu ion released from Cu containing material plays an important role. Cu ion might alter the structure of proteins [35], or disrupt enzyme structure and functions
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between the chemical composition, microstructure, mechanical property and antibacterial activity suggests that it should be possible to get comprehensive property by optimizing the chemical composition and microstructure. Also the fatigue property of Ti–Cu under biological environment needs to be assessed for clinical application. 5. Conclusion Ti–Cu alloys exhibited antibacterial property against E. coli and S. aureus. The Cu content affects the existence form of Cu in the Ti–Cu, the Cu ion release, and in turn influences the antibacterial activity seriously. The crucial Cu content in Ti–Cu alloys should be at least 5 wt.% in order to obtain stable and strong antibacterial activity against E. coli and S. aureus bacteria. The bacterial mechanism was thought to be related to the Cu ion release due to the galvanic corrosion between Cu-rich phase and titanium matrix. Fig. 11. Antibacterial rate of Ti–Cu alloys against E. coli and S. aureus with change in Cu content.
by binding to sulfur- or carboxylate-containing groups [36], or facilitate deleterious activity in superoxide radicals and cause “multiple hit damage” at target sites [37]. Our results also indicate that only Ti–Cu alloys with more than 5 wt.% Cu exhibits strong antibacterial activity, proving again that the antibacterial activity is really dependent on the Cu content. Fig. 12 illustrates the relationship between the antibacterial rate and the Cu ion concentration in the immersion solution. When the Cu ion concentration in the immersion solution is more than 0.036 mg/L, the antibacterial rate is larger than 99%, which is the recommended value for a strong antibacterial material [30]. Therefore, it is possible to predict the antibacterial property of Ti–Cu alloys by measuring the Cu ion release concentration in the immersion solution. In Ti–2.0 wt.% Cu, only Ti2Cu phase was formed and the Cu ion concentration kept at a low level, 0.0176 mg/L after 24 h immersion while the antibacterial rate is very low. In Ti–Cu alloys with 5 wt.% or more copper, Cu-rich phase besides Ti2Cu phase was formed and the Cu ion concentration kept at a relatively high level even after 24 h immersion, which responds to a high antibacterial rate. From the above results, it can be deduced that the Cu-rich phase plays a very important role in the antibacterial property of Ti–Cu alloys. The above results demonstrate that the Cu content in Ti–Cu sintered alloys seriously influences the existence form of Cu and the Cu ion release behavior, and in turn affects the mechanical property and the antibacterial property. In order to get strong and stable antibacterial property, the Cu content has to be at least 5 wt.% in Ti–Cu sintered alloy. However, the low ductility of Ti–Cu alloys will be a big problem for the clinical application. Above understanding on the relationship
Fig. 12. Relationship between the antibacterial rate and the Cu ion concentration in immersion solution.
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