Accepted Manuscript

Synthesis of silver–hydroxyapatite composite with improved antibacterial properties

Jianrong Wang, Xue Gong, Jun Hai, Tianrong Li

PII: S0042-207X(17)31672-X
DOI: 10.1016/j.vacuum.2018.03.015
Reference: VAC 7858

To appear in: Vacuum

Received Date: 22 November 2017

Revised Date: 26 February 2018
Accepted Date: 13 March 2018

Please cite this article as: Wang J, Gong X, Hai J, Li T, Synthesis of silver–hydroxyapatite composite
with improved antibacterial properties, Vacuum (2018), doi: 10.1016/j.vacuum.2018.03.015.

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 ACCEPTED MANUSCRIPT

Synthesis of silver–hydroxyapatite composite with improved

antibacterial properties

Jianrong Wanga,*, Xue Gongb, Jun Haic, Tianrong Lic,*

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a
Gansu Provincial Maternity and Child-care Hospital, Lanzhou 730050, P. R. China
b
Key Laboratory of Evidence of Science and Technology Research and Application, Gansu

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Institute of Political Science and Law, Lanzhou, 730070, P. R. China

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c
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province,

and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R.

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China AN
* Corresponding author.

E-mail address: wangjr_123@163.com (J. Wang), litr@lzu.edu.cn (T. Li).

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Abstract Hydroxyapatite is an important biomaterial for potential clinical applications. However,

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the lack of antibacterial activity restricts its application. To overcome this defect, here we prepared

a silver-incorporated hydroxyapatite composite, Ag–HA, and tested its antibacterial properties.

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Via the chemical precipitation of the suspension of chitosan, CaCl2 and NaH2PO4, an intermediate,

chitosan-hydroxyapatite composite CS–HA, was obtained. After the reduction of AgNO3 in

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CS–HA suspension by NaBH4, centrifugation and calcination, Ag–HA was collected. As the
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reference materials of Ag–HA, the hydroxyapatite paricle HA-2 was gained via the calcination of

CS–HA. To illustrate the role of chitosan in the preparation, the hydroxyapatite paricle HA-1 was

also synthesized with a similar method to HA-2 except for the use of chitosan. Powder X-ray

diffraction, X-ray photoelectron spectroscopy, fourier transform–infrared spectroscopy, and

scanning electron microscope were used to confirm and character Ag–HA, HA-1 and HA-2. We

found, by using chitosan, Ag–HA and HA-2 had more regular morphology than that of HA-1. The

antimicrobial activities of Ag–HA and HA-2 against Gram-positive Staphylococcus aureus and
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Gram-negative Escherichia coli were also evaluated via optical density tests. The results indicate

the incorporation of silver could endow hydroxyapatite particle with excellent antibacterial effect.

Keywords:Hydroxyapatite; Silver; Composite; Chitosan; Antibacterial property

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1. Introduction

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Deveolpment of man-made implant is crucial important to orthopedic and dental applications. In

recent years, carbon nanotubes [1], polymers [2], graphene-based substrates [3], zirconia based dental

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ceramics [4], magnesium phosphate cements [5], layered double hydroxides [6] and hydroxyapatite

[7,8] were researched as the major components of artificial bone, teeth or soft dental tissues. Among

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these materials, hydroxyapatite (HA, Ca10(PO4)6(OH)2) is the main mineral component of
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biological hard issues (such as bone and teeth) and constitutes about 70% by weight of the human

bone [9]. Different synthetic methods and biomedical applications of hydroxyapatite have
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received great attention because of its high bioactivity and osteo-conductive characteristics. Up to

now, abundant synthetic methods have been developed, such as sol-gel process, chemical
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precipitation, solid-state reaction, chemical vapor deposition, and reverse micro emulsion [7-11].
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Furthermore, hydroxyapatite was also researched as functionalized carrier for drug delivery

[12,13], tooth repair [14] and cartilage repair [15].

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However, the biocompatibility of hydroxyapatite is seriously influenced by the bacterial

proliferation on hydroxyapatite surface. Orthopaedic implant surfaces always provide ideal

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substrate to bacterial growth. As the result, bacterial infection is one of the most serious

complications of implant surgery, and it always leads to severe physiological damage and
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additional costly surgical procedures [16-18]. In order to provide antimicrobial activity to

hydroxyapatite, many approches were carried out. For example, the calcium hydroxyapatite

ceramic blocks have been impregnated by antibiotic [19]. Magnetized plasma with lower hybrid

resonance and excitation of helicon waves have been used to form crystalline thin films on

hydroxyapatite surface [20]. Hydroxyapatite/antibiotic composite coatings have also been

prepared via vacuum cold spraying [21]. Li et al. have used peptide-modified tannic acid to coat

on hydroxyapatite surface and found this composite could inhibit bacterial adhesion and activity in
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a long term [22]. Kurtjak et al. have incorporated of Ga3+ ions into hydroxyapatite to achieve effective

biological response [23].

Silver compounds and silver particles are well known for their broad spectrum antimicrobial

activity due to the release of silver ions [24-28]. Remarkably, silver particles have the advantage

of long term antimicrobial activity from a single use, and low probability of microorganism

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developing resistance. Plenty of composites contained silver particles have already shown obvious

antibacterial activities [29-31]. Moreover, many preparation methods of silver–hydroxyapatite

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composites and the researches on antimicrobial activities of these composites were also reported.

For example, Yatesa and co-workers deposited silver nanoparticles on hydroxyapatite coatings by

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a two-stage electrochemical synthesis method [32]. They found Staphylococcus aureus bacteria

growth was inhibited by coatings of Ag–hydroxyapatite, while hydroxyapatite coatings without

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silver had little effect on growth of the bacteria. Jegatheeswaran and Sundrarajan prepared
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hydroxyapatite/polyethylene glycol/Ag nanocomposite particles via a sol–gel route [33]. Their

results revealed silver nanoparticles loaded on the synthesized hydroxyapatite/polyethylene glycol

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showed a synergistic antibacterial effect against Gram-negative bacterium Escherichia coli. Due to

wet chemical precipitation method has the advantage of simple preparation, Gokcekaya and
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Webster used the precipitation method to synthesize Ag-incorporated hydroxyapatite [34].

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However, their preparation process was vague and the description of the phosphate used in their

reaction was incorrect.

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In order to invent a new and clear preparation method for silver–hydroxyapatite composite

via chemical precipitation method and verify the antimicrobial activities of the synthesized
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composite against both Gram-negative and Gram-positive bacteria, in this research, we

synthesized silver-incorporated hydroxyapatite composite (Ag–HA) and its reference materials

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without silver (namely HA-1 and HA-2) via precipitation method. Furthermore, we used scanning

electron microscope, fourier transform–infrared spectroscopy, powder X-ray diffraction and X-ray

photoelectron spectroscopy to confirm and character Ag–HA, HA-1, and HA-2. Finally, we

measured the antibacterial abilities of Ag–HA and HA-2 in vitro. Results showed Ag–HA

composite could effectively inhibit Gram-positive Staphylococcus aureus and Gram-negative

Escherichia coli, while the contrast, HA-2, has no inhibition against both of the bacteria. In sum,

this research provides a useful method to prepare silver-incorporated hydroxyapatite composite,

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and shows the incorporation of silver is an effective strategy to improve the antibacterial effect of

hydroxyapatite particles. It may promote the applications of hydroxyapatite in the orthopedic and

dental field.

2. Materials and methods

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2.1. Materials

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All chemicals were commercial and used without further purification. Sodium phosphate

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monobasic dihydrate (NaH2PO4.2H2O, Aladdin, 99.0%), calcium chloride dihydrate (CaCl2.2H2O,

Aladdin, 99.0%), sodium hydroxide (NaOH, Aladdin, 96%), chitosan (Aladdin), sodium

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borohydride (NaBH4, Sinopharm Chemical Reagent Co., Ltd., 96%) and silver nitrate (AgNO3,
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Macklin, 99.8%) were used as received. Deionized water was used in all experiments.
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2.2. Preparation of hydroxyapatite paricles (HA-1) without chitosan

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Hydroxyapatite paricles were synthesized according to a previously reported method [35]

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with a slight modification. Practically, NaH2PO4.2H2O (3.7435 g, 0.024 mol) and CaCl2.2H2O

(5.8804 g, 0.040 mol) were separately dissolved in 20 mL deionized water. The NaH2PO4 solution
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was slowly added to the CaCl2 solution under vigorous stirring. The Ca/P stoichiometric ratio was

1.67. The pH of the solution was adjusted to 10 with NaOH aqueous solution. After aging for 24 h,
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the precipitate was centrifuged, washed with deionized water, and fully dried in an oven. The

resulting solid was grinded with an agate mortar and calcined in a muffle furnace at 900 oC for 45
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min. Finally, the target product, hydroxyapatite paricle HA-1, was obtained.

2.3. Preparation of hydroxyapatite paricles (HA-2) with chitosan

A suspension of chitosan (CS) was fully mixed with CaCl2 solution (5.8804 g CaCl2.2H2O in

20 mL deionized water) under vigorous stirring. Then NaH2PO4 solution (3.7435 g

NaH2PO4.2H2O in 12 mL deionized water) was slowly added. The following pH adjustment, aging,
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centrifugalization, washing, and drying processes were same as that of HA-1. An intermediate,

CS–HA, was collected. After the grind and calcinations (at 900 oC for 45 min in a muffle furnace)

of the as-prepared CS–HA, the hydroxyapatite paricles HA-2 were gained.

2.4. Preparation of silver–hydroxyapatite composite (Ag–HA)

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The prepared CS–HA (0.3260 g) was dispersed in 15 mL deionized water. After sonication

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for 30 min, the suspension was cooled to 4 oC with an ice bath, and AgNO3 (0.0652g, 0.038 mmol)

was added in under vigorous stirring. Afterwards, NaBH4 solution (0.0462 g NaBH4 in 13 mL

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deionized water) was slowly added within 30 min. The reaction temperature should be no more

than 4 oC during that time. The suspension was centrifuged, washed with deionized water, and

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fully dried in an oven. The resulting product was grinded with agate mortar, and calcined in a
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muffle furnace at 900 oC for 45 min. Finally, the silver–hydroxyapatite composite Ag–HA was

obtained.
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2.5. Characterization
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The morphologies of the samples were examined with a scanning electron microscope (SEM,

TESCSN MIRA 3 XMU). Fourier transform–infrared spectroscopy (FT–IR) over the range of

400–4000 cm-1 was applied on a Thermo Mattson FT–IR spectrometer. Powder X-ray diffraction
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(PXRD) patterns were measured with a RigakuMiniFlex diffractometer using CuKα radiation at λ

= 1.5406 Å in the range of 20o≤ 2θ ≤ 60o. The Ag content in the silver–hydroxyapatite composite,
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Ag–HA, was determined by inductively coupled plasma optical emission spectrometer (Analytik
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Jena PQ 9000). X-ray photoelectron spectroscopy (XPS) spectra were generated by a PHI-5702

multifunctional X-ray photoelectron spectrometer. All optical measurements were performed at

room temperature under ambient conditions.

2.6. Antibacterial tests

According to a previous literature [36], Gram-positive Staphylococcus aureus (S.aureus) or

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Gram-negative Escherichia coli (E.coli) were grown in Muller-Hinton broth medium at 37 oC

to obtain isolated colonies. Then, the colonies were suspended in 0.85% sterile saline. The

turbidity was adjusted to McFarland 0.5 standard. The hybrid bacteria solution was incubated

at 37 oC on a shaking table after the addition of Ag–HA solution (or HA-2 solution). The

optical density at 600 nm was measured at defined time intervals of time for 600 minutes.

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The growth curves were obtained by plotting the optical density versus the incubation time.

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3. Results and discussion

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3.1. Synthesis of Ag–HA composite

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We obtained hydroxyapatite particle HA-1 with irregular morphology (Fig. 1a) via
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conventional chemical precipitation (without chitosan) and heat treatment at 900 oC. By using

chitosan (CS) as modifier during the chemical precipitation, hydroxyapatite particle HA-2
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was synthesized. The SEM micrograph shows HA-2 particles had more uniform morphology

and particle size (~3 µm) (Fig. 1b) than HA-1 particles. Nazeer et al. synthesized a series of
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intercalated chitosan/hydroxyapatite nanocomposite films. After thermal degradation of these

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films, small hydroxyapatite particles also could be formed. However, these particles were

fused together to form larger plate due to the heating temperature [9]. Chitosan is a derivative
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of chitin, which is mainly obtained by chitin deacetylation. They are polymorphous

noncrystalline or partly crystalline biopolymers. In previous studies, chitosan was usually

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used to form chitosan/hydroxyapatite film [9,37,38]. In this study, we found using chitosan as

modifier during wet chemical precipitation process could effectually improve the final
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morphology and particle size of hydroxyapatite particles.

In order to incorporate silver and hydroxyapatite particles, AgNO3 was reduced by NaBH4 in

the suspension of aforementioned CS–HA; then the resulting precipitate was centrifuged and

calcined. Finally, the silver-incorporated hydroxyapatite composite, Ag–HA, was gained. The

calcination temperature was selected to be 900 oC in our experiments, science the melting

temperature of silver is 961 oC [39] and higher temperature may promote the volatilization of

silver.
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From the SEM micrographs, we found the Ag–HA particles (Fig. 1c) were similar with HA-2

in geometric dimension, while the surface of Ag–HA was rough due to the deposition of Ag

particles. Based on inductively coupled plasma optical emission spectrometer (ICP–OES) analysis,

the content of silver in Ag–HA was found to be 6.6%.

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3.2. FT-IR analysis

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FT–IR spectra were used to gain the insights into the surface chemical structure of HA-1,

HA-2, and Ag–HA. In Fig. 2, the strong absorption bands at 1090 cm-1 and 1050 cm-1 are ascribed

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to the phosphate asymmetric stretching vibration (ν3) for these three samples [9,40]. The weak

absorption at 962 cm-1 is attributed to symmetric stretching (ν1) of P–O bond. The bands at 607

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cm-1 and 562 cm-1 are attributed to the bending vibrations of PO43-. The phosphate stretching mode
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ν2 appears at 474 cm−1. The broad absorbance at 3430 cm-1 and absorbance at 1630 cm-1 are

attributed to adsorbed and/or bound H2O of the samples. The characteristic IR bands that belong to
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β-tricalcium phosphate (940 and 1188 cm-1) and calcium pyrophosphate (725 and 1213 cm-1) [9]

were not observed in our FT–IR spectra. It indicates HA-1, HA-2, and Ag–HA have high purity.
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For Ag–HA, the molecular vibrations approximately correspond to a stoichiometric

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hydroxyapatite. This proves that silver did not enter into the apatite structure.
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3.3. PXRD analysis

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The phases and structures of HA-1, HA-2, and Ag–HA composite were studied via powder

X-ray diffraction (PXRD). The PXRD patterns of HA-1 (Fig. 3a) and HA-2 (Fig. 3b) coincided
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with pure hydroxyapatite phase with hexagonal crystal structure (JCPDS Card No. 9-432), and did

not show the existence of impure phase. Based on the PXRD patterns and FT–IR spectra of HA-1

and HA-2, we confirmed the phase formation of hydroxyapatite. The sharp diffraction peaks of

HA-2 indicate the crystallinity of HA-2 is better than that of HA-1. For Ag–HA composite,

besides the diffractions belonged to hydroxyapatite phase, additional silver phase was also

observed. The intense diffraction peaks at 2θ values of 38.1o and 44.3o (Fig. 3c) correspond to

metallic silver (JCPDS Card No. 4-0783) [29]. The crystal planes corresponding to the
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characteristic diffraction peaks of hydroxyapatite and silver were also marked in Fig. 3.

3.4. XPS analysis

X-ray photoelectron spectroscopy (XPS) characterization was performed to determine the

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chemical composition of the samples and identify the chemical state of silver element in Ag–HA.

As shown in Fig. 4a, three kinds of element contained in hydroxyapatite (i.e. Ca, O, and P) were

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all contained in HA-1, HA-2 and Ag–HA composite. Furthermore, the XPS high-resolution

spectra of the Ag3d state in Ag–HA showed Ag 3d5/2 peak and Ag 3d3/2 peak were obviously

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centered at 368.0 eV and 374.0 eV, respectively (Fig. 4b). According to literature [41], these two

peaks belong to metallic silver, which is in good agreement with the XRD analysis.

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3.5. Antibacterial activity

In order to test the antimicrobial activities of Ag–HA, antibacterial tests were carried out.
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Gram-positive S.aureus and Gram-negative E.coli were chosen as the model of bacteria. The

bacterial suspension alone was used as the positive control. Due to chitosan was used in the
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preparations of both HA-2 and Ag–HA, we chose HA-2 as the contrast sample of Ag–HA.
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The bacteria suspension (200 µL) was seeded in the solution of 0.002 g of Ag–HA (or HA-2)

in 20 mL of culture medium; then the resulting solution was incubated at 37 oC on a shaking table.
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The optical density at 600 nm (OD600) was measured at defined time intervals. The bacterial

growth can be indicated by using the OD600, since the growth of bacteria is proportional to the
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optical density of the bacteria suspension.

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The growth curves of E.coli and S.aureus cultured with Ag–HA (HA-2 or the positive

control) were shown in Fig. 5a and Fig. 5b, respectively. From the growth curves of the bacteria,

it can be seen the positive control group shows fastest S-sharp growth tendency for both E.coli and

S.aureus during the testing time (600 minutes). Notably, nearly no growth was observed for E.coli

and S.aureus suspensions in the presence of Ag–HA. For HA-2, the growth curves were similar to

that of the control group. It means HA-2, as the contrast sample of Ag–HA, have no inhibition

against both of the bacteria, and the antimicrobial activity of Ag–HA is due to the introduced

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silver. We confirm the incorporation of silver is a valid strategy to endow hydroxyapatite particles

with excellent antibacterial effect, and guess Ag–HA composite in this study may possess

potential in biomedicine applications.

4. Conclusions

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In summary, via the chemical precipitation of the suspension of chitosan, CaCl2 and NaH2PO4,

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an intermediate, chitosan-hydroxyapatite composite CS–HA, was collected. After the reduction of

AgNO3 in CS–HA suspension by NaBH4, centrifugation and calcination, the target product,

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silver–hydroxyapatite composite Ag–HA, was obtained. The hydroxyapatite paricle HA-2 was

gained via the calcination of CS–HA; and the hydroxyapatite paricle HA-1 was synthesized with a

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similar method to HA-2 except for the use of chitosan. As the result of using chitosan as the
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modifier in precipitation process, Ag–HA and HA-2 particles had more uniform morphology and

particle size than HA-1. Ag–HA possessed high phase purity; and silver element existed in
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elemental silver state and distributed as grains. Moreover, Ag–HA expressed excellent inhibiting

bacteria growth effect on S.aureus and E.coli in vitro; nevertheless, the contrast sample, HA-2, did
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not obviously show antibacterial property. The incorporation of silver with hydroxyapatite is an
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effective strategy to improve the antibacterial effect of hydroxyapatite. Silver ion (Ag+) has strong

broad spectrum of antibacterial activity. However, it is still a controversial issue as to whether it

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can show the desired antibacterial activity while being toxic to mammalian cells [42]. The detailed

relationship between silver concentration in the composite and antibacterial efficacy and
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cytotoxicity deserves to be studied in future researches.

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Acknowledgments This work was financially supported by Lanzhou Science and Technology

Project (Grant No. 2016-3-122).

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Fig. 1 SEM micrographs of (a) HA-1 particles, (b) HA-2 particles, and (c) Ag–HA composite.

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Fig. 2 FT–IR spectra of (a) HA-1 particles, (b) HA-2 particles, and (c) Ag–HA composite.
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Fig. 3 PXRD patterns of (a) HA-1 particles, (b) HA-2 particles, and (c) Ag–HA composite.
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Fig. 4 (a) XPS survey spectra of the sample ((1) HA-1 particles, (2) HA-2 particles, and (3)
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Ag–HA composite), and (b) XPS high-resolution spectra of the Ag3d state in Ag–HA composite.
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Fig. 5 Growth curves of (a) Gram-negative Escherichia coli and (b) Gram-positive Staphylococcus

aureus cultured with Ag–HA (2 mg) or HA-2 (2 mg) in the culture medium system.
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Highlights:

New synthetic approach for preparation of Ag–hydroxyapatite composite.

Efficient inactivation of S. aureus and E. coli over the synthesized Ag–hydroxyapatite.

Incorporation of metallic silver improved the antibacterial effect of hydroxyapatite particles.

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