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Synthesis of silver-incorporated hydroxyapatite nanocomposites for antimicrobial implant coatings
Applied Surface Science 273 (2013) 748–757
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Synthesis of silver-incorporated hydroxyapatite nanocomposites for antimicrobial implant coatings Xiangmei Liu a,b , Yanan Mou b , Shuilin Wu b , H.C. Man a,∗ a
Department of Industrial and Systems Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, School of Materials Science and Engineering, Hubei University, Wuhan 430062, PR China b
a r t i c l e
i n f o
Article history: Received 22 January 2013 Accepted 27 February 2013 Available online 7 March 2013 Keywords: Hydroxyapatite Nanocomposite Silver Antibacterial Implant
a b s t r a c t Because of excellent osteoconductivity and resorbability, hydroxyapatite (HA) is commonly used as a bone substitute material or implant coating. Both ionic and metallic silver are considered to have a broad spectrum of antimicrobial properties especially associated with biomaterial-related infections. The present work proposes a facile chemical reduction method to synthesize an Ag incorporated HA nanocomposite. Ammoniacal silver solution was firstly prepared and then added into the HA solution, followed by hydrazine hydrate (N2 H4 ·H2 O) being used to reduce the silver ions to metallic silver. The formed Ag nanoparticles had diameters of 20–30 nm and were firmly attached on the HA particle surfaces. This approach can also keep the integrity of the HA chemical structure and the morphology. The strain Escherichia coli was used to evaluate the antibacterial effect of the nanocomposite. An In vitro bacterial adhesion study indicated a significant enhancement in the antibacterial property of silver containing HA. © 2013 Published by Elsevier B.V.
1. Introduction Besides the similarity of chemical compositions of hydroxyapatite (HA, Ca10 (PO4 )6 (OH)2 ) and related calcium phosphates to the basic mineral phase of natural bone, the bioactivity, dissolution range, and resorption properties are also very close to those of natural bone. Therefore, the synthetic forms of these calcium phosphates are extensively used as implant materials for the reconstruction and substitution of hard tissues [1–5]. As a biocompatible and osteoconductive biomaterial, HA has the capability of encouraging bone formation and in providing direct chemical bonding with natural bone. It is also widely applied as a coating material on prostheses to improve their biological properties [6] and different coating methods have been explored [7–11]. However, proteins, amino acids, and other organic substances are easily adsorbed on the HA surface, which in turn favors the adsorption and replication of the bacteria on the HA and subsequently induces implant-related infections [12]. Once implant-related infections occur, the implant becomes loosened due to the bacterial colony formation [13], the bone healing process becomes more complicated and potentially devastating complications such as implant failure, the need for multiple operations, and sometimes even amputation may result.
∗ Corresponding author. Tel.: +852 27666629; fax: +852 23624741. E-mail address: [email protected] (H.C. Man). 0169-4332/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsusc.2013.02.130
Hydroxyapatite containing antimicrobial agents is expected to prevent or cure implant-associated infections by directly releasing antimicrobial agents to local regions such as the implant tissue interface. Some studies have demonstrated that antibiotics can be easily loaded as a coating on implant surfaces [14,15]. However, there are concerns over the efficacy of antibiotics in the face of the growing problem of antibiotic resistant bacteria and also the short term effect of the antibiotics [16]. Therefore, as an inorganic antibacterial agent, silver has gained more attention due to its broad-spectrum antibacterial properties at very low concentrations, good biocompatibility, satisfactory safety in usage and inherent stability [17–19]. Silver exhibits strong antibacterial effects through interacting with the proteins and enzymes of bacteria and causes structural damage to the cell wall and bacterial membrane. Specifically, silver binds to bacterial DNA and RNA and thereby prevents bacterial reproduction. Also, silver binds to sulfhydryl groups of metabolic enzymes and thereby inhibits the electron transport chain of the cell, which results in bacterial destruction [20]. Therefore, silver containing HA composites combining osseointegration of HA with the antibacterial effect of Ag, have great potential application in bone substitute materials or in coating metal implants to prevent implant-related infections. One aspect of silver containing HA composite material is that Ag ions are incorporated into the HA structure through ion-exchange methods such as sol–gel or coprecipitation routes. The calcium is replaced by silver resulting in a Ca-deficient hydroxyapatite
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Fig. 1. The surface morphologies of nano HA–Ag composite powders (a) as-received HA; (a1) high magnification image of (a); (b) HA–0.5% Ag; (c) HA–1% Ag; (d) HA–2% Ag; (e) HA–5% Ag; (e1) high magnification image of (e).
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Fig. 2. The EDS patterns of nano HA–Ag composite powders (a) as-received HA; (b) HA–0.5% Ag; (c) HA–1% Ag; (d) HA–2% Ag; (e) HA–5% Ag.
[21,22]. Although the slow-rate release of Ag ions from the HA structure can provide an antibacterial effect, the depletion of calcium could have negative effects on the structural stability of HA and its osteoconduction ability [12,23,24]. Another aspect is that Ag particles or nanoparticles attach to HA surfaces and then take charge of antibacterial activity. Some authors described related preparation methods, such as the sol–gel method, chemical reduction by the OH− groups from the surface of the HA, magnetron cosputtering, and the colloidal chemical route followed by a thermal treatment [25–28]. Here, a very simple reduction method is described to obtain an Ag containing HA nanocomposite where silver is attached on HA surface. Commercially purchased nano HA powders were used as starting materials. Ammoniacal silver solution was firstly prepared and then added into the HA solution, followed by hydrazine hydrate (N2 H4 ·H2 O) being dropwise added to reduce the silver ions to metallic silver. In this process, no other phase or impurities were introduced. Different silver content composites were easily prepared. The structure, morphology, vibrational and optical properties of Ag–HA were systematically characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and UV–vis spectroscopy. All the results provided evidence that the silver reduction process had no impact on the parent HA structure and morphologies. The antibacterial experiment revealed that the nano composite had its own super antibacterial effect. The current research proposes a favorable use of such composites for bone reconstruction or for a surface coating on prostheses. 2. Experimental procedures 2.1. Material preparation Five compositions of HA were prepared by a very simple reduction method: HA, HA loaded by 0.5 wt% Ag, 1 wt%Ag,
2 wt% Ag, 5 wt% Ag. The procedures are described in detail as follows. The used hydroxyapatite (HA) powders were purchased from Aladdin Reagent Corporation with an average particle size of about 100 nm (purity > 99.5%, BET), with AgNO3 crystals as the silver source. After precise calculation, the materials were weighed using a FR-300 MKI electronic balance with a precision of 0.1 mg. Firstly, the AgNO3 solution was dropped into a clean beaker and 30% ammonia was then added until the initial precipitate just dissolved. Secondly, as-received HA powders were dissolved by deionized water and then subjected to ultrasound for 30 min after adding the dispersing agent (PVP). Thirdly, the HA solution was then stirred in a magnetic stirrer 1 h. In the fourth step, the silver ammonia solution was dropwise added into the HA solution. Subsequently, hydrazine hydrate (N2 H4 ) aqueous solution was added into the mixture to reduce the silver ions into metallic silver. During the reduction reaction, the white HA powders gradually changed to gray color. After completion of the reaction, the nano Ag-containing HA powders were collected through filtration, rinsed with deionized water and alcohol several times and finally oven dried at 60 ◦ C. Composites with different amounts of silver content could be obtained by adjusting the amount of AgNO3 solution. 2.2. Materials characterization 2.2.1. Scanning electron microscopy (SEM) The morphology of Ag–HA powder was examined by scanning electron microscopy (SEM, JEOL JSM6510LV and JSM820). The elemental contents of the nanocomposite were determined by energy dispersive X-ray spectroscopy (EDS). 2.2.2. Transmission electron microscopy (TEM) TEM studies were carried out using a JEOL 200 CX. The specimen for TEM imaging was prepared by suspending the particles in alcohol. A drop of well dispersed supernatant was placed on
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Fig. 3. The Ag distribution in nano HA–Ag composite powders by elemental mapping (a) HA–0.5% Ag; (b) HA–1% Ag; (c) HA–2% Ag; (e) HA–5% Ag.
a 200 mesh copper grid, followed by drying the sample at ambient conditions before it was attached to the sample holder of the microscope. The microstructure and composition of the nanocomposite were analyzed by TEM and selected area electron diffraction (SAED), respectively. 2.2.3. X-ray diffraction (XRD) XRD was conducted using a Siemens D500 X-ray diffractomer with a copper K␣ X-ray source operated at 40 kV and 30 mA. The spectra were acquired in the 2 range of 10–80◦ with step increments of 0.05◦ and a count time of 0.5 s per step. The XRD patterns were studied based on the JCPC standard diffraction database using the software MDI JAD5.0.
2.2.4. Fourier transform infrared spectroscopy (FT-IR) FT-IR analysis of the samples was undertaken on a FT-IR spectrometer (Perkin-Bhaskar-Elmer Co., USA). 1% of the powder was mixed and ground with 99% KBr and the powder mixtures were pressed at load to form 10 diameter tablets. The FTIR measurements were taken in the range of 400–4000 cm−1 . 2.2.5. UV–vis spectroscopy UV–vis spectroscopy was performed using a UV-3600 UV–vis spectrophotometer and the examination range was 350–800 nm. 2.2.6. Antimicrobial test Antibacterial action of Ag–HA powder was studied by the spread plate method. One milligram of selected Ag–HA (0,
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Fig. 4. TEM images of as-received HA powders (a) aggregated HA particles; (b) high-resolution TEM images; (c) SAED pattern; and (d) EDS pattern.
2, 5% Ag–HA,) was mixed individually with 1 mL of phosphate buffer solution (pH 7.4), containing 1 × 105 cells/mL Escherichia coli, in flat-bottom test tubes. Each tube was shaken at 200 rpm, at 37 ◦ C, for 24 h. One hundred microliters of shaken Ag–HA solution were placed on petri plates in duplicate and 15 mL of molten tryptose soy agar was overlaid onto the inoculum, spread evenly and left undisturbed for the agar to solidify. These plates, after solidification, were incubated at 37 ◦ C C for 24 h. The colony formation was observed and photographed.
3. Results 3.1. Morphologies of Ag–HA nanocomposite powders Fig. 1 shows the surface morphologies of nano HA–Ag composite powders. All the powders, including as-received HA and silver containing HA powders, were in the nano scale and aggregated together loosely. The high magnification SEM images of selected powders such as pure HA (Fig. 1a1), and HA–5% Ag (Fig. 1e1) revealed that the aggregation was not compact. It suggested that the silver incorporation processing did not change the original morphology and distribution of HA nano powders. The EDS patterns of the nano Ag–HA composite powders are shown in Fig. 2. The elemental composition of the powders was mainly O, P, Ca and Ag, which means that Ag has been successfully incorporated into HA powders. In the following courses, we examined the silver distribution in Ag–HA nanocomposites by elemental mapping as shown in Fig. 3. Generally, the silver particles were evenly distributed in the entire powders. With the increase of silver incorporation content, the Ag signals in the elemental mapping gradually became stronger, especially for 5% Ag containing HA (Fig. 3e).
3.2. Microstructures and phase analysis Fig. 4 shows the microstructure and chemical composition of pure HA nanoparticles. The HA clusters are composed of some short HA sheets in a width range of 20–30 nm and a length round 60–70 nm. In addition, the HA clusters are loosely aggregated, which is consistent with the observation of SEM. The SAED pattern exhibits typical polycrystalline features. The polycrystals are composed of some small single crystals because some light diffraction spots coexist with some polycrystal rings in the selected area. The EDS pattern shown in Fig. 4c indicates that the mainly compositional elements are Ca, P and O. A small amount of C was also observed due to the dissolution of CO2 gas during the producing process. The Cu signal from this copper mesh and Si was possibly contaminated during TEM sample preparation. The microstructure and chemical composition of 2% Ag–HA composites are shown in Fig. 5. Some black Ag nanoparticles appeared, as shown in Fig. 5a. The produced Ag nanoparticles are of spherical shape, with a diameter range of 20–30 nm. The majority of silver nanoparticles attached on the HA particles surface and some Ag nanoparticles exist among the HA particles. The SAED pattern shown in Fig. 5b indicates that the HA powders still remain polycrystalline. The EDS pattern shown in Fig. 5c indicates that the main compositional elements are Ca, P, O, C and Ag. Fig. 6 shows the TEM images and elemental analysis of a 5% Ag–containing HA composite. It exhibits the same phenomenon as that shown in Fig. 5. The particle size has no obvious difference for the two types of nanocomposites. The increase of silver content did not result in the formation of large silver particles, but the latter showed a stronger silver signal in the EDS pattern (Fig. 6c), indicating more Ag in the latter. The elemental quantity analysis of the above three types of powders by EDS is shown in Table 1. It also confirmed that silver particles were successfully loaded into the HA powders, and the silver content increased as the starting content of AgNO3 increased.
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Fig. 5. TEM images of HA–2% Ag powders (a) aggregated particles; (b) SAED pattern; and (c) EDS pattern.
Fig. 7 shows the UV–vis absorption spectra taken from the solution of HA and several Ag containing HA powders. The change in optical features shown in these spectra is in good accord with the previous observations by SEM and TEM. There are no peaks
for HA. For 2% Ag–HA spectra, the appeared plasmon peak that appears at about 410 nm indicates the formation of Ag nanoparticles with diameters of 20–30 nm. The intensity of this plasmon peak increased for 5% Ag–HA, indicating a higher silver concentration
Fig. 6. TEM images of HA–5% Ag powders without dispersing agent (a) aggregated particles; (b) SAED pattern; and (c) EDS pattern.
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Table 1 The EDS analysis of nano particles shown in TEM images (at.%). Spectrum
P
Ca
Ag
HA HA–2% Ag HA–5% Ag
38.80 36.44 36.86
61.20 57.17 52.96
0.00 6.40 10.18
1# 2# 3# 4# 5#
0.30
Absorbance
0.25 0.20
1#
0.15
5#
0.10
4# 3#
HA HA-0.5%Ag HA-1%Ag HA-2%Ag HA-5%Ag
2#
0.05
Fig. 9. XRD patterns of HA powders and Ag–HA nanocomposites.
0.00 300
400
500
600
3#
and 1091 cm−1 . Three types of OH− band modes were traced in the spectra. The stretching mode of OH− ions was observed at 3570, 3434 cm−1 due to the presence of an organized water structure in HA. The bending mode of OH group at 1629 cm−1 belonged to the H2 O molecules. The libration mode for OH− at 632 cm−1 was the structural band of HA. All the results were very similar to those reported in the literature [26]. The main features of Ag–HA composite spectra are also shown in the curves. The intensity, position and shape of peaks were very similar to those of the parent HA nanopowders. No new peaks were found in these curves. Fig. 9 shows the phase compositions of HA powder and Ag–HA nanocomposites identified by XRD. Although the Ag signal was not strong due to its small amount, some related peaks could still be recognized, like (1 1 1), (2 0 0), (2 2 0) and (3 1 1), which could be indexed to the structure of cubic silver (Ag 3C) (JCPDS no. 04-0783). Fig. 10 exhibits the significant difference in antibacterial effect between the pure HA and Ag incorporated HA nanocomposites. After 24 h incubation, both blank control and pure HA plates were fully covered with E. coli colonies, as shown in Fig. 10(a) and (b). Moreover, pure HA formed more E. coli colonies than the control plate. Ag incorporated HA nano composites inhibited the bacteria growth, as shown in Fig. 10(c)–(f). The numbers of E. coli colonies decreased with increasing silver content. Dozens of E. coli colonies were observed after 24 h incubation in the plate with 0.5% Ag–HA powders. However there was no bacterial colony formation for 1%, 2% and 5% Ag–HA nanocomposites, which demonstrate the complete antibacterial properties of these content nanocomposites.
4#
4. Discussion
5#
The simple chemical reduction method was successfully used to synthesize Ag containing HA nanocomposites. The reduced silver nanoparticles attached on the HA surface have no influence on the structure and morphology of the as-received HA powders. The formation mechanism of silver nanoparticles is revealed by Eqs. (1)–(3).
700
800
Wavelength (nm) Fig. 7. UV–vis absorption spectra of HA powders and Ag–HA nanocomposites.
[29]. Meanwhile, the peak position did not shift to longer wavelengths, which means the same particle size is the same between 5% Ag–HA and 2% Ag–HA. The results are consistent with the results of TEM. Because of the low Ag concentration, there are no obvious Ag signals for 0.5% Ag–HA and 1% Ag–HA spectra. Fig. 8 is the FT-IR spectra of HA powders and the Ag–HA nanocomposites. FT-IR spectroscopy was used to investigate the functional groups present in the HA and Ag–HA powders. The spectra of pure HA show the typical bands of carbonated HA [28]. A carbonate peak is observed because a part of the PO4 3− groups are substituted by carbonate ions, possibly formed by the dissolution of CO2 gas during the producing process [29]. The existence of C has been detected to be EDS of TEM. The related peaks at 876, 1421, and 1453 cm−1 correspond to the vibrations of CO3 2− . The vibration bands of PO4 3− appeared at 473, 565, 602, 962, 1035,
1#
500
1000
1500
1# 2# 3# 4# 5# 2000
HA HA-0.5%Ag HA-1%Ag HA-2%Ag HA-5%Ag 2500
3000
3436 3570
1453
1638
1421
876 1035 1091
565 602
962
473
Transitance (a.u.)
2#
3500
-1
Wavenumber (cm ) Fig. 8. FT-IR spectra of HA powders and Ag–HA nanocomposites.
4000
AgNO3 + NH3 · H2 O = 1 Ag2 O + 2NH3 2
1 Ag2 O + NH4 NO3 2
+ 12 H2 O
(1)
· H2 O = Ag(NH3 )2 OH + 32 H2 O
(2)
4Ag(NH3 )2 + + 4OH− + N2 H4 = 4Ag ↓ +N2 ↑ +8NH3 + 4H2 O
(3)
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Fig. 10. Representative photos of E. coli colonies on (a) blank as a control (b) HA nano powder; (c) 0.5% Ag–HA nanocomposite; (d) 1% Ag–HA nanocomposite; (e) 2% Ag–HA nanocomposite; (f) 5% Ag–HA nanocomposite.
Firstly, Ag ions in AgNO3 with NH3 ·H2 O formed complex compounds following Eqs. (1) and (2). Secondly, the complex compound reacted with hydrazine hydrate (N2 H4 ) to form metallic silver as shown in Eq. (3). According to this equation, except for the deposited Ag, the reaction products were all in gaseous states such as N2 . Because the reaction system was constantly stirred, the silver crystals could be dispersed into the entire solution
and attached on the HA surface once they are formed. Therefore, the aggregation of Ag nanoparticles was limited [31]. This approach did not introduce any other phase or impurity to the final Ag–HA composite. In addition, the merit of this approach is that through a one step reaction, metallic Ag is obtained. As reported in the literature [24], a similar composite was obtained. However, NaOH should be used to adjust the pH value with the product
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AgO, which needs to be deducted by Ar/H2 at higher temperature [24]. From the SEM, the pure HA kept its original morphologies, despite the incorporation of silver, which was very consistent with some published reports [26]. The EDS mapping revealed that the silver nanoparticles have a uniform distribution in the entire HA powder, which was compatible with the co-precipitation method [6]. TEM images confirmed that the formed Ag was of spherical shape and attached on the HA particles. During the reaction process, the silver did not replace any calcium in the HA, and the HA particles maintained their chemical structure integrity. The calcium is, however, replaced by silver, resulting in a Ca-deficient hydroxyapatite [21,22]. The depletion of calcium can induce negative effects on the structural stability of HA and impair its osteoconductive ability [12,23,24]. According to the TEM results in the literature [6,12], once the calcium in the HA is partly replaced by adding silver, no obvious black Ag nanoparticles appear in the TEM images. The UV–vis spectra displayed the typical spherical Ag nanoparticle feature, a plasmon peak at about 410 nm. The absence of higherwavelength features indicated a lack of anisotropy of the silver nanoparticles, in good agreement with the TEM images. Reported the plasmon peaks have been identified at 420 nm or 410 nm [30,31]. The smaller silver particles induced lower peak positions. A few researchers observed a broad plasmon peak at about 450 nm, which was attributed to the aggregated Ag particles [24]. Here, this TEM results showed that the silver nanoparticles were well dispersed and the particle size was in the range of 20–30 nm. Therefore, the plasmon peak was narrow and the peak position was close to those of reported results. Moreover, among five types of nanocomposites, the amount of silver nanoparticles was too low to get a strong signal from both the 0.5% and 1% Ag–HA samples. The FT-IR data revealed the main functional groups of PO4 3− , CO3 2− and OH in pure HA and Ag–HA nanocomposites. The incorporation of Ag did not induce obvious variations of the above peaks and the formation of new peaks. Ag O bonding from the AgO phase or Ag2 O phase was not found in the FTIR spectra, which indicated direct formation of metallic silver. Usually, the structural OH− group in the HA at 632 cm−1 is responsible for the reduction of silver ions and thereby favors the binding with HA particles [25,28]. The spectra in this work showed that the OH− bonding shape and position have no obvious change. Therefore, the OH− bond in pure HA was not involved in the reduction process. From the XRD patterns, the peaks at 38, 44.4 and 65.8 ◦ C were consistent with (1 1 3), (2 0 0), and (3 1 1) Bragg reflections, respectively, of the silver nanoparticles relating to the FCC structure. This confirmed that the silver ions were reduced to silver nanocrystals on the surface of HA, as seen in Fig. 1(a). There was no decrease in the intensity and broadening of the diffraction of the HA, indicating that the structure and crystals were not affected by the formation of the composite. The antibacterial results revealed that pure nano HA powder has no antibacterial property. On the contrary, the pure HA plate exhibited more bacterial colonies than the control sample. This formed the viewpoint that proteins, amino acids, and other organic substances are easily adsorbed on the HA surface, which could favor the adsorption and replication of the bacteria on the HA, usually inducing implant-related infections [12]. Therefore, the addition of the antibacterial properties of HA is necessary. After adding the antibacterial elements of Ag, Ag–HA nanocomposites exhibited completely strong antibacterial properties when the silver content reached 1%. This kind of antibacterial property was also reported in the literature but the threshold of the silver content was diverse. For example, it was revealed that silver nanoparticles at a concentration of 10 and 50 g/cm3 inhibited bacterial growth of 62% and 88%, respectively, while a concentration of 100 g/cm3 caused 100% inhibition of bacterial growth [24]. The
research reported that bacterial adhesion was significantly reduced on Ca10−x Agx (PO4 )6 (OH)2 samples with x = 0.2, when compared to samples with x = 0, but no significant difference in bacterial adhesion was observed between the different concentrations of Ag:HAp nanopowders [6]. The report found that all three samples (0.5, 1.0, and 1.5 AgHA) showed complete inhibition of the growth of bacteria under incubation conditions for 24 h and even after 48 h [12]. However the research were afforded very simple and visible evidence that the antibacterial effect of Ag–HA strongly depends on the silver content. 5. Conclusions In the current work, the team successfully loaded silver nanoparticles on HA surfaces by a facile chemical reduction method. The silver nanoparticles were stably and evenly distributed in the HA nanoparticles. The whole reaction process has no obvious effect on the structure and morphology of HA. When the silver content was higher than 2%, the UV–vis detected a very strong plasmon signal of silver nanoparticles. The FTIR spectra have no obvious difference for pure HA and Ag–HA, while the EDS, TEM and XRD results confirmed the formation of Ag. Preliminary in vitro antibacterial tests indicated that the novel Ag–HA nanocomposites exhibit high efficiency in killing bacteria. Higher silver content composites completely inhibited bacterial growth. Combined with the good biocompatibility of HA, the present approach provides a convenient method for the development of novel biomaterials with good biocompatibility and excellent antibacterial properties. Acknowledgments This work was jointly supported by The Hong Kong Polytechnic University Postdoctoral Fellowship Scheme (G-YX4Z), National Natural Science Foundation of China Nos. 50901032, 51101053, 81271715. References [1] M. Tirrell, E. Kokkoli, M. Biesalski, The role of surface science in bioengineered materials, Surface Science 500 (2002) 61–83. [2] L.L. Hench, Bioceramics Journal of the American Ceramic Society 81 (1998) 1705–1728. [3] Z. Shen, E. Adolfsson, M. Nygren, L. Gao, H. Kawaoka, K. Niihara, Dense hydroxyapatite-zirconia ceramic composites with high strength for biological applications, Advanced Materials 13 (2001) 214–216. [4] R.A.M.J. Claessens, Z.I. Kolar, Affinity of tin(II) and tin(II) diphosphonates for hydroxyapatite: an experimental and model study, Langmuir 16 (2000) 1360–1367. [5] W.P. McConnell, J.P. Novak, L.C. Brousseau III, R.R. Fuierer, R.C. Tenent, D.L. Feldheim, Electronic and optical properties of chemically modified metal nanoparticles and molecularly bridged nanoparticle arrays, Journal of Physical Chemistry B 104 (2000) 8925–8930. [6] C.S. Ciobanu, F. Massuyeau, L.V. Constantin, D. Predoi, Structural and physical properties of antibacterial Ag-doped nano-hydroxyapatite synthesized at 100 ◦ C, Nanoscale Research Letters 6 (2011) 1–8. [7] M.H. Wong, F.T. Cheng, H.C. Man, Characteristics, apatite forming ability and corrosion resistance of NiTi surface modified by AC anodization, Applied Surface Science 253 (2007) 7527–7534. [8] C.T. Kwok, P.K. Wong, F.T. Cheng, H.C. Man, Characterization and corrosion behavior of HA coatings on Ti6 Al4 V fabricated by electrophoretic deposition, Applied Surface Science 255 (2009) 6736–6744. [9] S. Yang, W. Xing, H.C. Man, Pulsed laser deposition of hydroxyapatite film on laser gas nitriding NiTi substrate, Applied Surface Science 255 (2009) 9889–9892. [10] Y. Bai, I.S. Park, S.J. Lee, One step approach for hydroxyapatite incorporated TiO2 coating on Ti via a combined technique of micro-arc oxidation and electrophoretic deposition, Applied Surface Science 257 (2011) 7010–7018. [11] R.A. Surmenev, A review of plasma assisted methods for calcium phosphate based coatings fabrication, Surface and Coatings Technology 206 (2012) 2035–2056. [12] N. Rameshbabu, T.S. Sampath Kumar, T.G. Prabhakar, V.S. Sastry, K.V.G.K. Murty, K. Prasad Rao, Antibacterial nanosized silver substituted hydroxyapatite: synthesis and characterization, Journal of Biomedical Materials Research Part A 80 (2007) 581–591.
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Synthesis of silver-incorporated hydroxyapatite nanocomposites for antimicrobial implant coatings Xiangmei Liu a,b , Yanan Mou b , Shuilin Wu b , H.C. Man a,∗ a
Department of Industrial and Systems Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, School of Materials Science and Engineering, Hubei University, Wuhan 430062, PR China b
a r t i c l e
i n f o
Article history: Received 22 January 2013 Accepted 27 February 2013 Available online 7 March 2013 Keywords: Hydroxyapatite Nanocomposite Silver Antibacterial Implant
a b s t r a c t Because of excellent osteoconductivity and resorbability, hydroxyapatite (HA) is commonly used as a bone substitute material or implant coating. Both ionic and metallic silver are considered to have a broad spectrum of antimicrobial properties especially associated with biomaterial-related infections. The present work proposes a facile chemical reduction method to synthesize an Ag incorporated HA nanocomposite. Ammoniacal silver solution was firstly prepared and then added into the HA solution, followed by hydrazine hydrate (N2 H4 ·H2 O) being used to reduce the silver ions to metallic silver. The formed Ag nanoparticles had diameters of 20–30 nm and were firmly attached on the HA particle surfaces. This approach can also keep the integrity of the HA chemical structure and the morphology. The strain Escherichia coli was used to evaluate the antibacterial effect of the nanocomposite. An In vitro bacterial adhesion study indicated a significant enhancement in the antibacterial property of silver containing HA. © 2013 Published by Elsevier B.V.
1. Introduction Besides the similarity of chemical compositions of hydroxyapatite (HA, Ca10 (PO4 )6 (OH)2 ) and related calcium phosphates to the basic mineral phase of natural bone, the bioactivity, dissolution range, and resorption properties are also very close to those of natural bone. Therefore, the synthetic forms of these calcium phosphates are extensively used as implant materials for the reconstruction and substitution of hard tissues [1–5]. As a biocompatible and osteoconductive biomaterial, HA has the capability of encouraging bone formation and in providing direct chemical bonding with natural bone. It is also widely applied as a coating material on prostheses to improve their biological properties [6] and different coating methods have been explored [7–11]. However, proteins, amino acids, and other organic substances are easily adsorbed on the HA surface, which in turn favors the adsorption and replication of the bacteria on the HA and subsequently induces implant-related infections [12]. Once implant-related infections occur, the implant becomes loosened due to the bacterial colony formation [13], the bone healing process becomes more complicated and potentially devastating complications such as implant failure, the need for multiple operations, and sometimes even amputation may result.
∗ Corresponding author. Tel.: +852 27666629; fax: +852 23624741. E-mail address: [email protected] (H.C. Man). 0169-4332/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsusc.2013.02.130
Hydroxyapatite containing antimicrobial agents is expected to prevent or cure implant-associated infections by directly releasing antimicrobial agents to local regions such as the implant tissue interface. Some studies have demonstrated that antibiotics can be easily loaded as a coating on implant surfaces [14,15]. However, there are concerns over the efficacy of antibiotics in the face of the growing problem of antibiotic resistant bacteria and also the short term effect of the antibiotics [16]. Therefore, as an inorganic antibacterial agent, silver has gained more attention due to its broad-spectrum antibacterial properties at very low concentrations, good biocompatibility, satisfactory safety in usage and inherent stability [17–19]. Silver exhibits strong antibacterial effects through interacting with the proteins and enzymes of bacteria and causes structural damage to the cell wall and bacterial membrane. Specifically, silver binds to bacterial DNA and RNA and thereby prevents bacterial reproduction. Also, silver binds to sulfhydryl groups of metabolic enzymes and thereby inhibits the electron transport chain of the cell, which results in bacterial destruction [20]. Therefore, silver containing HA composites combining osseointegration of HA with the antibacterial effect of Ag, have great potential application in bone substitute materials or in coating metal implants to prevent implant-related infections. One aspect of silver containing HA composite material is that Ag ions are incorporated into the HA structure through ion-exchange methods such as sol–gel or coprecipitation routes. The calcium is replaced by silver resulting in a Ca-deficient hydroxyapatite
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Fig. 1. The surface morphologies of nano HA–Ag composite powders (a) as-received HA; (a1) high magnification image of (a); (b) HA–0.5% Ag; (c) HA–1% Ag; (d) HA–2% Ag; (e) HA–5% Ag; (e1) high magnification image of (e).
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Fig. 2. The EDS patterns of nano HA–Ag composite powders (a) as-received HA; (b) HA–0.5% Ag; (c) HA–1% Ag; (d) HA–2% Ag; (e) HA–5% Ag.
[21,22]. Although the slow-rate release of Ag ions from the HA structure can provide an antibacterial effect, the depletion of calcium could have negative effects on the structural stability of HA and its osteoconduction ability [12,23,24]. Another aspect is that Ag particles or nanoparticles attach to HA surfaces and then take charge of antibacterial activity. Some authors described related preparation methods, such as the sol–gel method, chemical reduction by the OH− groups from the surface of the HA, magnetron cosputtering, and the colloidal chemical route followed by a thermal treatment [25–28]. Here, a very simple reduction method is described to obtain an Ag containing HA nanocomposite where silver is attached on HA surface. Commercially purchased nano HA powders were used as starting materials. Ammoniacal silver solution was firstly prepared and then added into the HA solution, followed by hydrazine hydrate (N2 H4 ·H2 O) being dropwise added to reduce the silver ions to metallic silver. In this process, no other phase or impurities were introduced. Different silver content composites were easily prepared. The structure, morphology, vibrational and optical properties of Ag–HA were systematically characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and UV–vis spectroscopy. All the results provided evidence that the silver reduction process had no impact on the parent HA structure and morphologies. The antibacterial experiment revealed that the nano composite had its own super antibacterial effect. The current research proposes a favorable use of such composites for bone reconstruction or for a surface coating on prostheses. 2. Experimental procedures 2.1. Material preparation Five compositions of HA were prepared by a very simple reduction method: HA, HA loaded by 0.5 wt% Ag, 1 wt%Ag,
2 wt% Ag, 5 wt% Ag. The procedures are described in detail as follows. The used hydroxyapatite (HA) powders were purchased from Aladdin Reagent Corporation with an average particle size of about 100 nm (purity > 99.5%, BET), with AgNO3 crystals as the silver source. After precise calculation, the materials were weighed using a FR-300 MKI electronic balance with a precision of 0.1 mg. Firstly, the AgNO3 solution was dropped into a clean beaker and 30% ammonia was then added until the initial precipitate just dissolved. Secondly, as-received HA powders were dissolved by deionized water and then subjected to ultrasound for 30 min after adding the dispersing agent (PVP). Thirdly, the HA solution was then stirred in a magnetic stirrer 1 h. In the fourth step, the silver ammonia solution was dropwise added into the HA solution. Subsequently, hydrazine hydrate (N2 H4 ) aqueous solution was added into the mixture to reduce the silver ions into metallic silver. During the reduction reaction, the white HA powders gradually changed to gray color. After completion of the reaction, the nano Ag-containing HA powders were collected through filtration, rinsed with deionized water and alcohol several times and finally oven dried at 60 ◦ C. Composites with different amounts of silver content could be obtained by adjusting the amount of AgNO3 solution. 2.2. Materials characterization 2.2.1. Scanning electron microscopy (SEM) The morphology of Ag–HA powder was examined by scanning electron microscopy (SEM, JEOL JSM6510LV and JSM820). The elemental contents of the nanocomposite were determined by energy dispersive X-ray spectroscopy (EDS). 2.2.2. Transmission electron microscopy (TEM) TEM studies were carried out using a JEOL 200 CX. The specimen for TEM imaging was prepared by suspending the particles in alcohol. A drop of well dispersed supernatant was placed on
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Fig. 3. The Ag distribution in nano HA–Ag composite powders by elemental mapping (a) HA–0.5% Ag; (b) HA–1% Ag; (c) HA–2% Ag; (e) HA–5% Ag.
a 200 mesh copper grid, followed by drying the sample at ambient conditions before it was attached to the sample holder of the microscope. The microstructure and composition of the nanocomposite were analyzed by TEM and selected area electron diffraction (SAED), respectively. 2.2.3. X-ray diffraction (XRD) XRD was conducted using a Siemens D500 X-ray diffractomer with a copper K␣ X-ray source operated at 40 kV and 30 mA. The spectra were acquired in the 2 range of 10–80◦ with step increments of 0.05◦ and a count time of 0.5 s per step. The XRD patterns were studied based on the JCPC standard diffraction database using the software MDI JAD5.0.
2.2.4. Fourier transform infrared spectroscopy (FT-IR) FT-IR analysis of the samples was undertaken on a FT-IR spectrometer (Perkin-Bhaskar-Elmer Co., USA). 1% of the powder was mixed and ground with 99% KBr and the powder mixtures were pressed at load to form 10 diameter tablets. The FTIR measurements were taken in the range of 400–4000 cm−1 . 2.2.5. UV–vis spectroscopy UV–vis spectroscopy was performed using a UV-3600 UV–vis spectrophotometer and the examination range was 350–800 nm. 2.2.6. Antimicrobial test Antibacterial action of Ag–HA powder was studied by the spread plate method. One milligram of selected Ag–HA (0,
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Fig. 4. TEM images of as-received HA powders (a) aggregated HA particles; (b) high-resolution TEM images; (c) SAED pattern; and (d) EDS pattern.
2, 5% Ag–HA,) was mixed individually with 1 mL of phosphate buffer solution (pH 7.4), containing 1 × 105 cells/mL Escherichia coli, in flat-bottom test tubes. Each tube was shaken at 200 rpm, at 37 ◦ C, for 24 h. One hundred microliters of shaken Ag–HA solution were placed on petri plates in duplicate and 15 mL of molten tryptose soy agar was overlaid onto the inoculum, spread evenly and left undisturbed for the agar to solidify. These plates, after solidification, were incubated at 37 ◦ C C for 24 h. The colony formation was observed and photographed.
3. Results 3.1. Morphologies of Ag–HA nanocomposite powders Fig. 1 shows the surface morphologies of nano HA–Ag composite powders. All the powders, including as-received HA and silver containing HA powders, were in the nano scale and aggregated together loosely. The high magnification SEM images of selected powders such as pure HA (Fig. 1a1), and HA–5% Ag (Fig. 1e1) revealed that the aggregation was not compact. It suggested that the silver incorporation processing did not change the original morphology and distribution of HA nano powders. The EDS patterns of the nano Ag–HA composite powders are shown in Fig. 2. The elemental composition of the powders was mainly O, P, Ca and Ag, which means that Ag has been successfully incorporated into HA powders. In the following courses, we examined the silver distribution in Ag–HA nanocomposites by elemental mapping as shown in Fig. 3. Generally, the silver particles were evenly distributed in the entire powders. With the increase of silver incorporation content, the Ag signals in the elemental mapping gradually became stronger, especially for 5% Ag containing HA (Fig. 3e).
3.2. Microstructures and phase analysis Fig. 4 shows the microstructure and chemical composition of pure HA nanoparticles. The HA clusters are composed of some short HA sheets in a width range of 20–30 nm and a length round 60–70 nm. In addition, the HA clusters are loosely aggregated, which is consistent with the observation of SEM. The SAED pattern exhibits typical polycrystalline features. The polycrystals are composed of some small single crystals because some light diffraction spots coexist with some polycrystal rings in the selected area. The EDS pattern shown in Fig. 4c indicates that the mainly compositional elements are Ca, P and O. A small amount of C was also observed due to the dissolution of CO2 gas during the producing process. The Cu signal from this copper mesh and Si was possibly contaminated during TEM sample preparation. The microstructure and chemical composition of 2% Ag–HA composites are shown in Fig. 5. Some black Ag nanoparticles appeared, as shown in Fig. 5a. The produced Ag nanoparticles are of spherical shape, with a diameter range of 20–30 nm. The majority of silver nanoparticles attached on the HA particles surface and some Ag nanoparticles exist among the HA particles. The SAED pattern shown in Fig. 5b indicates that the HA powders still remain polycrystalline. The EDS pattern shown in Fig. 5c indicates that the main compositional elements are Ca, P, O, C and Ag. Fig. 6 shows the TEM images and elemental analysis of a 5% Ag–containing HA composite. It exhibits the same phenomenon as that shown in Fig. 5. The particle size has no obvious difference for the two types of nanocomposites. The increase of silver content did not result in the formation of large silver particles, but the latter showed a stronger silver signal in the EDS pattern (Fig. 6c), indicating more Ag in the latter. The elemental quantity analysis of the above three types of powders by EDS is shown in Table 1. It also confirmed that silver particles were successfully loaded into the HA powders, and the silver content increased as the starting content of AgNO3 increased.
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Fig. 5. TEM images of HA–2% Ag powders (a) aggregated particles; (b) SAED pattern; and (c) EDS pattern.
Fig. 7 shows the UV–vis absorption spectra taken from the solution of HA and several Ag containing HA powders. The change in optical features shown in these spectra is in good accord with the previous observations by SEM and TEM. There are no peaks
for HA. For 2% Ag–HA spectra, the appeared plasmon peak that appears at about 410 nm indicates the formation of Ag nanoparticles with diameters of 20–30 nm. The intensity of this plasmon peak increased for 5% Ag–HA, indicating a higher silver concentration
Fig. 6. TEM images of HA–5% Ag powders without dispersing agent (a) aggregated particles; (b) SAED pattern; and (c) EDS pattern.
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Table 1 The EDS analysis of nano particles shown in TEM images (at.%). Spectrum
P
Ca
Ag
HA HA–2% Ag HA–5% Ag
38.80 36.44 36.86
61.20 57.17 52.96
0.00 6.40 10.18
1# 2# 3# 4# 5#
0.30
Absorbance
0.25 0.20
1#
0.15
5#
0.10
4# 3#
HA HA-0.5%Ag HA-1%Ag HA-2%Ag HA-5%Ag
2#
0.05
Fig. 9. XRD patterns of HA powders and Ag–HA nanocomposites.
0.00 300
400
500
600
3#
and 1091 cm−1 . Three types of OH− band modes were traced in the spectra. The stretching mode of OH− ions was observed at 3570, 3434 cm−1 due to the presence of an organized water structure in HA. The bending mode of OH group at 1629 cm−1 belonged to the H2 O molecules. The libration mode for OH− at 632 cm−1 was the structural band of HA. All the results were very similar to those reported in the literature [26]. The main features of Ag–HA composite spectra are also shown in the curves. The intensity, position and shape of peaks were very similar to those of the parent HA nanopowders. No new peaks were found in these curves. Fig. 9 shows the phase compositions of HA powder and Ag–HA nanocomposites identified by XRD. Although the Ag signal was not strong due to its small amount, some related peaks could still be recognized, like (1 1 1), (2 0 0), (2 2 0) and (3 1 1), which could be indexed to the structure of cubic silver (Ag 3C) (JCPDS no. 04-0783). Fig. 10 exhibits the significant difference in antibacterial effect between the pure HA and Ag incorporated HA nanocomposites. After 24 h incubation, both blank control and pure HA plates were fully covered with E. coli colonies, as shown in Fig. 10(a) and (b). Moreover, pure HA formed more E. coli colonies than the control plate. Ag incorporated HA nano composites inhibited the bacteria growth, as shown in Fig. 10(c)–(f). The numbers of E. coli colonies decreased with increasing silver content. Dozens of E. coli colonies were observed after 24 h incubation in the plate with 0.5% Ag–HA powders. However there was no bacterial colony formation for 1%, 2% and 5% Ag–HA nanocomposites, which demonstrate the complete antibacterial properties of these content nanocomposites.
4#
4. Discussion
5#
The simple chemical reduction method was successfully used to synthesize Ag containing HA nanocomposites. The reduced silver nanoparticles attached on the HA surface have no influence on the structure and morphology of the as-received HA powders. The formation mechanism of silver nanoparticles is revealed by Eqs. (1)–(3).
700
800
Wavelength (nm) Fig. 7. UV–vis absorption spectra of HA powders and Ag–HA nanocomposites.
[29]. Meanwhile, the peak position did not shift to longer wavelengths, which means the same particle size is the same between 5% Ag–HA and 2% Ag–HA. The results are consistent with the results of TEM. Because of the low Ag concentration, there are no obvious Ag signals for 0.5% Ag–HA and 1% Ag–HA spectra. Fig. 8 is the FT-IR spectra of HA powders and the Ag–HA nanocomposites. FT-IR spectroscopy was used to investigate the functional groups present in the HA and Ag–HA powders. The spectra of pure HA show the typical bands of carbonated HA [28]. A carbonate peak is observed because a part of the PO4 3− groups are substituted by carbonate ions, possibly formed by the dissolution of CO2 gas during the producing process [29]. The existence of C has been detected to be EDS of TEM. The related peaks at 876, 1421, and 1453 cm−1 correspond to the vibrations of CO3 2− . The vibration bands of PO4 3− appeared at 473, 565, 602, 962, 1035,
1#
500
1000
1500
1# 2# 3# 4# 5# 2000
HA HA-0.5%Ag HA-1%Ag HA-2%Ag HA-5%Ag 2500
3000
3436 3570
1453
1638
1421
876 1035 1091
565 602
962
473
Transitance (a.u.)
2#
3500
-1
Wavenumber (cm ) Fig. 8. FT-IR spectra of HA powders and Ag–HA nanocomposites.
4000
AgNO3 + NH3 · H2 O = 1 Ag2 O + 2NH3 2
1 Ag2 O + NH4 NO3 2
+ 12 H2 O
(1)
· H2 O = Ag(NH3 )2 OH + 32 H2 O
(2)
4Ag(NH3 )2 + + 4OH− + N2 H4 = 4Ag ↓ +N2 ↑ +8NH3 + 4H2 O
(3)
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Fig. 10. Representative photos of E. coli colonies on (a) blank as a control (b) HA nano powder; (c) 0.5% Ag–HA nanocomposite; (d) 1% Ag–HA nanocomposite; (e) 2% Ag–HA nanocomposite; (f) 5% Ag–HA nanocomposite.
Firstly, Ag ions in AgNO3 with NH3 ·H2 O formed complex compounds following Eqs. (1) and (2). Secondly, the complex compound reacted with hydrazine hydrate (N2 H4 ) to form metallic silver as shown in Eq. (3). According to this equation, except for the deposited Ag, the reaction products were all in gaseous states such as N2 . Because the reaction system was constantly stirred, the silver crystals could be dispersed into the entire solution
and attached on the HA surface once they are formed. Therefore, the aggregation of Ag nanoparticles was limited [31]. This approach did not introduce any other phase or impurity to the final Ag–HA composite. In addition, the merit of this approach is that through a one step reaction, metallic Ag is obtained. As reported in the literature [24], a similar composite was obtained. However, NaOH should be used to adjust the pH value with the product
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AgO, which needs to be deducted by Ar/H2 at higher temperature [24]. From the SEM, the pure HA kept its original morphologies, despite the incorporation of silver, which was very consistent with some published reports [26]. The EDS mapping revealed that the silver nanoparticles have a uniform distribution in the entire HA powder, which was compatible with the co-precipitation method [6]. TEM images confirmed that the formed Ag was of spherical shape and attached on the HA particles. During the reaction process, the silver did not replace any calcium in the HA, and the HA particles maintained their chemical structure integrity. The calcium is, however, replaced by silver, resulting in a Ca-deficient hydroxyapatite [21,22]. The depletion of calcium can induce negative effects on the structural stability of HA and impair its osteoconductive ability [12,23,24]. According to the TEM results in the literature [6,12], once the calcium in the HA is partly replaced by adding silver, no obvious black Ag nanoparticles appear in the TEM images. The UV–vis spectra displayed the typical spherical Ag nanoparticle feature, a plasmon peak at about 410 nm. The absence of higherwavelength features indicated a lack of anisotropy of the silver nanoparticles, in good agreement with the TEM images. Reported the plasmon peaks have been identified at 420 nm or 410 nm [30,31]. The smaller silver particles induced lower peak positions. A few researchers observed a broad plasmon peak at about 450 nm, which was attributed to the aggregated Ag particles [24]. Here, this TEM results showed that the silver nanoparticles were well dispersed and the particle size was in the range of 20–30 nm. Therefore, the plasmon peak was narrow and the peak position was close to those of reported results. Moreover, among five types of nanocomposites, the amount of silver nanoparticles was too low to get a strong signal from both the 0.5% and 1% Ag–HA samples. The FT-IR data revealed the main functional groups of PO4 3− , CO3 2− and OH in pure HA and Ag–HA nanocomposites. The incorporation of Ag did not induce obvious variations of the above peaks and the formation of new peaks. Ag O bonding from the AgO phase or Ag2 O phase was not found in the FTIR spectra, which indicated direct formation of metallic silver. Usually, the structural OH− group in the HA at 632 cm−1 is responsible for the reduction of silver ions and thereby favors the binding with HA particles [25,28]. The spectra in this work showed that the OH− bonding shape and position have no obvious change. Therefore, the OH− bond in pure HA was not involved in the reduction process. From the XRD patterns, the peaks at 38, 44.4 and 65.8 ◦ C were consistent with (1 1 3), (2 0 0), and (3 1 1) Bragg reflections, respectively, of the silver nanoparticles relating to the FCC structure. This confirmed that the silver ions were reduced to silver nanocrystals on the surface of HA, as seen in Fig. 1(a). There was no decrease in the intensity and broadening of the diffraction of the HA, indicating that the structure and crystals were not affected by the formation of the composite. The antibacterial results revealed that pure nano HA powder has no antibacterial property. On the contrary, the pure HA plate exhibited more bacterial colonies than the control sample. This formed the viewpoint that proteins, amino acids, and other organic substances are easily adsorbed on the HA surface, which could favor the adsorption and replication of the bacteria on the HA, usually inducing implant-related infections [12]. Therefore, the addition of the antibacterial properties of HA is necessary. After adding the antibacterial elements of Ag, Ag–HA nanocomposites exhibited completely strong antibacterial properties when the silver content reached 1%. This kind of antibacterial property was also reported in the literature but the threshold of the silver content was diverse. For example, it was revealed that silver nanoparticles at a concentration of 10 and 50 g/cm3 inhibited bacterial growth of 62% and 88%, respectively, while a concentration of 100 g/cm3 caused 100% inhibition of bacterial growth [24]. The
research reported that bacterial adhesion was significantly reduced on Ca10−x Agx (PO4 )6 (OH)2 samples with x = 0.2, when compared to samples with x = 0, but no significant difference in bacterial adhesion was observed between the different concentrations of Ag:HAp nanopowders [6]. The report found that all three samples (0.5, 1.0, and 1.5 AgHA) showed complete inhibition of the growth of bacteria under incubation conditions for 24 h and even after 48 h [12]. However the research were afforded very simple and visible evidence that the antibacterial effect of Ag–HA strongly depends on the silver content. 5. Conclusions In the current work, the team successfully loaded silver nanoparticles on HA surfaces by a facile chemical reduction method. The silver nanoparticles were stably and evenly distributed in the HA nanoparticles. The whole reaction process has no obvious effect on the structure and morphology of HA. When the silver content was higher than 2%, the UV–vis detected a very strong plasmon signal of silver nanoparticles. The FTIR spectra have no obvious difference for pure HA and Ag–HA, while the EDS, TEM and XRD results confirmed the formation of Ag. Preliminary in vitro antibacterial tests indicated that the novel Ag–HA nanocomposites exhibit high efficiency in killing bacteria. Higher silver content composites completely inhibited bacterial growth. Combined with the good biocompatibility of HA, the present approach provides a convenient method for the development of novel biomaterials with good biocompatibility and excellent antibacterial properties. Acknowledgments This work was jointly supported by The Hong Kong Polytechnic University Postdoctoral Fellowship Scheme (G-YX4Z), National Natural Science Foundation of China Nos. 50901032, 51101053, 81271715. References [1] M. Tirrell, E. Kokkoli, M. Biesalski, The role of surface science in bioengineered materials, Surface Science 500 (2002) 61–83. [2] L.L. Hench, Bioceramics Journal of the American Ceramic Society 81 (1998) 1705–1728. [3] Z. Shen, E. Adolfsson, M. Nygren, L. Gao, H. Kawaoka, K. Niihara, Dense hydroxyapatite-zirconia ceramic composites with high strength for biological applications, Advanced Materials 13 (2001) 214–216. [4] R.A.M.J. Claessens, Z.I. Kolar, Affinity of tin(II) and tin(II) diphosphonates for hydroxyapatite: an experimental and model study, Langmuir 16 (2000) 1360–1367. [5] W.P. McConnell, J.P. Novak, L.C. Brousseau III, R.R. Fuierer, R.C. Tenent, D.L. Feldheim, Electronic and optical properties of chemically modified metal nanoparticles and molecularly bridged nanoparticle arrays, Journal of Physical Chemistry B 104 (2000) 8925–8930. [6] C.S. Ciobanu, F. Massuyeau, L.V. Constantin, D. Predoi, Structural and physical properties of antibacterial Ag-doped nano-hydroxyapatite synthesized at 100 ◦ C, Nanoscale Research Letters 6 (2011) 1–8. [7] M.H. Wong, F.T. Cheng, H.C. Man, Characteristics, apatite forming ability and corrosion resistance of NiTi surface modified by AC anodization, Applied Surface Science 253 (2007) 7527–7534. [8] C.T. Kwok, P.K. Wong, F.T. Cheng, H.C. Man, Characterization and corrosion behavior of HA coatings on Ti6 Al4 V fabricated by electrophoretic deposition, Applied Surface Science 255 (2009) 6736–6744. [9] S. Yang, W. Xing, H.C. Man, Pulsed laser deposition of hydroxyapatite film on laser gas nitriding NiTi substrate, Applied Surface Science 255 (2009) 9889–9892. [10] Y. Bai, I.S. Park, S.J. Lee, One step approach for hydroxyapatite incorporated TiO2 coating on Ti via a combined technique of micro-arc oxidation and electrophoretic deposition, Applied Surface Science 257 (2011) 7010–7018. [11] R.A. Surmenev, A review of plasma assisted methods for calcium phosphate based coatings fabrication, Surface and Coatings Technology 206 (2012) 2035–2056. [12] N. Rameshbabu, T.S. Sampath Kumar, T.G. Prabhakar, V.S. Sastry, K.V.G.K. Murty, K. Prasad Rao, Antibacterial nanosized silver substituted hydroxyapatite: synthesis and characterization, Journal of Biomedical Materials Research Part A 80 (2007) 581–591.
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