Porous titanium scaffold surfaces modified with silver loaded gelatin microspheres and their antibacterial behavior

    Porous titanium scaffold surfaces modified with silver loaded gelatin microspheres and their antibacterial behavior Mengting Li, Yi Wang, Lili Gao, Yuhua Sun, Jianxin Wang, Shuxin Qu, Ke Duan, Jie Weng, Bo Feng PII: DOI: Reference:

S0257-8972(15)30437-0 doi: 10.1016/j.surfcoat.2015.12.006 SCT 20762

To appear in:

Surface & Coatings Technology

Received date: Revised date: Accepted date:

14 September 2015 24 November 2015 3 December 2015

Please cite this article as: Mengting Li, Yi Wang, Lili Gao, Yuhua Sun, Jianxin Wang, Shuxin Qu, Ke Duan, Jie Weng, Bo Feng, Porous titanium scaffold surfaces modified with silver loaded gelatin microspheres and their antibacterial behavior, Surface & Coatings Technology (2015), doi: 10.1016/j.surfcoat.2015.12.006

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ACCEPTED MANUSCRIPT Porous Titanium Scaffold Surfaces Modified with Silver Loaded Gelatin Microspheres and Their Antibacterial Behavior

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Mengting Li1, Yi Wang, Lili Gao, Yuhua Sun, Jianxin Wang, Shuxin Qu, Ke Duan,

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Jie Weng, Bo Feng *

Key Laboratory of Advanced Technology for Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu

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610031, China

Author information *Corresponding author: Bo Feng Tel: +86 028 87634023; Fax: +86 28 87601371. E-mail addresses: fengbo@ swjtu.edu.cn (Bo Feng).

ACCEPTED MANUSCRIPT Abstract Recent years, porous titanium (PT) attracts much attention because of its growing application as medical implants. However, bacterial infections related to implants

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remain one of the most common and serious complications. In this study, silver loaded gelatin microspheres (Ag/GMSs) were fabricated and incorporated into porous

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titanium to get antibacterial implants. Prior to incorporating microspheres, oxide film and micro/nano structures were formed on porous titanium by micro-arc oxidation (MAO) to improve its activity, and the resulted sample is named MPT. Samples were

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characterized with scanning electron microscope, energy dispersive X-ray spectroscope, transmission electron microscope, X-ray diffractometer, laser scattering

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particle analyzer and atomic absorption spectrometer. The results showed that Ag0 particles were loaded to the gelatin microspheres with average diameter of 4.46 µm

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and Ag/GMSs distributed uniformly on the pore walls of PT and MPT. Ag0 particles

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were demonstrated not only formed on the surface of gelatin microspheres but also inside them. The silver loaded samples exhibited a high antibacterial ability against

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both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The MPT sample was not only better in supporting osteoblast cell viability and good cell proliferation, but also was beneficial to immobilize the silver loaded microspheres to

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achieve a stronger antibacterial effect than PT. The MPT sample with silver loaded gelatin microspheres increased the cumulative release period of Ag and reached to more than ten days. The main idea of this study also elicited a new surface functionalization strategy for improving antibacterial ability of porous titanium. Key words: porous titanium; surface coating modification; silver loaded gelatin microspheres; antibacterial property; osteoblast

1. Introduction Because of its excellent mechanical properties, corrosion resistance and biocompatibility, titanium becomes an ideal choice for the long-term replacement of hard tissue and has been widely used in biomedical engineering. Porous titanium was introduced due to its superior biocompatibility, adjustable mechanical properties and

ACCEPTED MANUSCRIPT porous structure [1, 2]. Porous structure not only can improve the fixation of solid implants to the surrounding bone tissue by allowing high cell seeding density and tissue in-growth, but also can benefit body fluids being transported through the porous

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implant [3, 4]. However, it is known that titanium implants are generally bioinert. Susceptible surface suffers from problems of interfacial stability with host tissues. So,

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a bioactive surface with osteoconductive property is required to prevent the formation of fibrous tissue and avoid isolating them from the surrounding bone. Surface modification by micro-arc oxidation (MAO) can alter chemical composition, surface

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roughness, and morphology of materials [5-9]. Both oxide film and micro/nano structures formed on PT are in favor of the adhesion, proliferation and osteogenesis of

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the osteoblast-like cells in vitro [10-13] and enhance osteointegration of Ti implants in vivo [14]. However, another serious dilemma presented for hard tissue replacement

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materials is lack of antimicrobial property, and deep infection becomes the most

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common complication. Bacterial infection may occur at the time of surgery or derive from bacteria from remote sources where bacteria are seeded at the vicinities of the

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implants [15]. Since the impacted bone graft is initially an avascular area, systemic antimicrobial drug cannot easily achieve access to the graft site whereby the risk of postoperative infection is heightened. Local antibiotic delivery has, therefore, become

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a much-researched area in recent years [16]. The three-dimensional scaffold plays a critical role in the delivery process of growth factors and some drugs. Most of the previous drug delivery works focused on that they can be encapsulated or imbedded within the porous matrices like the three-dimensional scaffold and delivered in a sustained manner to enhance cell growth and morphogenesis, leading to a functionally organized tissue [3, 17, 18]. Gelatin is a denatured and biodegradable protein obtained by the acid and alkaline processing of collagen. The biosafety of gelatin microspheres has been proven through its long clinical usage in surgical biomaterials and as an ingredient in drugs [19, 20]. In recent years, gelatin microspheres (GMSs) are widely employed as a delivery vehicle for the controlled release of biomolecules due to its ability to form polyion complexes with charged therapeutic compounds such as proteins, nucleotides and polysaccharides to prevent rapid release at first and realize a

ACCEPTED MANUSCRIPT subsequent sustained release [21-23]. And it has been used for the controlled release of various kinds of drugs, such as antibiotic drug gentamycin sulfate [24], antihypertensive drugs nifedipine [25], antiasthmatic drug theophylline (THP) [26],

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anti-inflammatory drugs diclofenac sodium salt [27]and ceftiofur [19].

A large amount of antibiotics like cephalothin, carbenicillin, amoxicillin,

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cefamandol, tobramycin, and vancomycin have been incorporated in bone implants to form a local release system to prevent post-surgical infections favoring early osteointegration stage [28-30]. However, different drugs have different targeted. For

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example, vancomycin can kill only Gram-positive bacteria and is ineffective against Gram-negative bacteria. However, tobramycin-resistant can be more sensitive on

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Gram-negative bacteria. This study possibly investigated antibacterial effects to against Gram-positive and Gram-negative two kinds of bacteria [31]. Silver has long

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been known to be a potent antibacterial agent and exhibit more merits than these

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drugs when doped into implants to overcome the microbial contaminant and infectious disease. For example, silver has the greatest potential against both

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Gram-positive and Gram-negative bacteria [30, 31]. Silver incorporating can efficiently inhibit bacterial attachment onto biomaterials and kill them [32]. In this study, porous titanium scaffolds were treated by the MAO to form the

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oxide layer and micro/nano structures. The gelatin mirospheres as silver carrier were incorporated into the porous titanium scaffold uniformly. With the degradation of the gelatin microspheres, silver ions released from the microspheres at a controllable rate. The oxide film layer and micro/nano structures not only benefit osteoblast attachment and proliferation, but also can provide more favorable conditions for immobilization of the silver loaded microspheres. In this way, antibacterial efficiency increased with higher silver content in the porous titanium and the duration time of antimicrobial was prolonged.

2. Materials and methods

2.1. Preparation of porous titanium (PT)

ACCEPTED MANUSCRIPT Porous titanium scaffolds were prepared by powder metallurgy sintering technology. Ammonium hydrogen carbonate (NH4HCO3) particles in the range of 200–300 μm were chosen as spacer material. The titanium powders and NH4HCO3

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particles (1:1) were uniformly mixed and pressed into green compacts under a

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pressure of 60 MPa. Subsequently, the green compacts were heat-treated at 180 ℃

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for 3 h to volatilize the spacer and form cancellous structure. Finally, the samples were sintered at 1200 ℃ for 2 h in a vacuum of 1.0×10−3 Pa. All of the reagents were

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of analytical grade in this work including the following experiments.

2.2 Surface modification of porous titanium by micro-arc oxidation

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All the prepared PT scaffolds were cleaned with acetone, ethyl alcohol and deionized water, successively. And then, the sample was used as anode and a graphite

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plate as cathode in an electrolytic bath with 0.1 M H2SO4 solution. During this

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treatment, a DC voltage of 110 V was applied for 3 mins and temperature of the solution was controlled by a cooling system. MAO treatment resulted in the formation

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of the oxide layer and micro/nano porous titanium scaffold, namely MPT.

2.3 Synthesis of gelatin microspheres

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Gelatin microspheres (GMSs) were prepared by a water-in-oil (w/o) emulsion approach. Briefly, 10 wt% gelatin (type A) was preheated and then dropwisely added into 60mL of light liquid paraffin (10:60, w/w) containing 3% (w/w) Span-80 at 60 ℃under constant stirring for 30 min. When the w/o emulsion system was cooled to 4 ℃, glutaraldehyde was added slowly and stirred for 2 h, followed by the addition of 30 mL acetone. The resulted microspheres were washed by ethanol and isopropanol to remove residual light liquid paraffin, collected by filtration and preserved after freeze-drying.

2.4 Preparation of silver loaded gelatin microspheres After the prepared GMSs were soaked in silver nitrate solution (10-3 M) for 12 h, 4×10-3 M sodium borohydride solution was dropwisely added into the solution and

ACCEPTED MANUSCRIPT aged for 2 h. The silver particles loaded gelatin microspheres (Ag/GMSs) were washed by centrifugation, and protected from light. The preparation process is shown

2.5 Preparation of Ag/GMSs loaded porous titanium

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in Scheme1

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Ag/GMSs were dispersed into ethanol under constant stirring and then the porous titanium samples (PT and MPT) were immersed in the suspension for 12 hours. The samples with Ag/GMSs were taken out and cleaned, and noted Ag/GMSs/PT and

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2.6 Characterization of samples

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Ag/GMSs/MPT respectively.

The morphologies of samples were observed by scanning electron microscopy

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(SEM, JEOL-JSM-700IF). The crystalline structure of PT and the phase components

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of MPT were analyzed by X-ray diffraction (XRD, Phlips X’Pert PRO) using a Cu Kα radiation in the regular range 2θ=20~70◦ at the scanning speed of 5◦/min. Particle size

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analyzer was utilized to measure the particle size of GMSs. X-ray photoelectron spectroscopy (XPS, Kratos XSAM-800, Al Kα radiation) was used to determine the chemical composition and the valence of elements. The microstructure of GMSs was

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characterized by transmission electron microscopy (TEM, Jeol, Germany). To detect whether silver particles formed inside the network structure of GMSs, Ag/GMSs were embedded in polymethyl methacrylate and sectioned with a diamond band saw (Leica 1600, Germany). The Ag/GMSs section was observed by TEM, and the Ag crystal structure type was additionally verified by careful analysis of high resolution transmission electron micrographs (HR-TEM). The chemical composition was also detected by energy dispersive X-ray spectroscopy (EDS, FEI Quanta 200).

2.7 In vitro the release of Ag+ The Ag/GMSs/PT and Ag/GMSs/MPT samples were respectively immersed in 10 mL of phosphate buffer solution (PBS) in a test tube, and incubated under constant

ACCEPTED MANUSCRIPT shaking at 37 ℃. At designated time points, 10 mL solution was taken out for Ag concentration measurement, and 10 mL of fresh PBS was added to the original tube. This process was carried out for a total of 15 days to determine the Ag release

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dynamics. The Ag concentration in PBS was measured with atomic absorption

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spectrometer (AAS, TAS-990).

2.8 Cell viability assay

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Sd rat’s osteoblasts were primary-cultured, maintained in Dulbecco's modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS), 1%

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antibiotic/antimycotic mixture in an incubator supplied with 5% CO2 at 37 ℃ in humidity and passaged to the 4th generation prior to use. The cells were seeded at a density of 5 × 104/mL on test samples and control materials (PT). AlamarBlueTM

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assay was performed to evaluate the cell proliferation and metabolic activity

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according to alamarBlue cell viability assay protocol. After osteoblasts were incubated with GMSs/PT, GMSs/MPT, Ag/GMSs/PT and Ag/GMSs/MPT for 1, 3 and

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5 days, the samples were rinsed three times with PBS, and then 1mL of the fresh medium containing 10 vol% alamarBlue (Gibco, Invitrogen) was added into each well

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and incubated at 37 ℃. 4 h later, 200 μL of the medium in each well was collected to detect optical density (OD) at 570 nm and the absorbance value was proportional to the cell viability. The samples were fixed with 2.5% glutaraldehyde and kept at 4 ℃ overnight. After being washed twice with PBS, all the samples were stained with 4’, 6-iamidino-2-henylindo (DAPI) for 10 min and then visualized by fluorescence microscopy (FV1000, Olympus, Japan).

2.9 Antibacterial performance assay The antibacterial activity of Ag/GMSs/PT and Ag/GMSs/MPT scaffolds was investigated to against 109 CFU mL-1 E. coli (ATCC 25922) as a model of Gram-negative bacterium and 107 CFU mL-1 S. aureus (ATCC 6538) as a model of Gram-positive bacterium. All the samples were sterilized with 75% alcohol for six

ACCEPTED MANUSCRIPT hours and rinsed three times with sterile PBS. The samples were placed on 12-well culture plates and 4 mL of bacterial-containing medium (109 CFU mL-1) was added into each well. After incubation for 1, 3 and 5 days, 200 µL bacterial-containing

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medium in each well was collected to assess the antimicrobial efficiency of various samples. On 5 days, all the samples were fixed with 2.5% glutaraldehyde (GA). A

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sequential dehydration of 50%, 70%, 80%, 90%, 95%, and 100% ethanol was performed, followed by drying overnight. Then samples were sputter coated with platinum prior to imaging under the SEM

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2.10 Analysis of statistical

All the obtained results were expressed as mean ± standard and were assessed

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statistically using one-way analysis of variance (ANOVA). Statistical significance was determined and accepted at *p<0.05 and obvious significance was determined and

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accepted at **p<0.01.

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3. Results and Discussion

3.1 Surface treatment

Fig. 1a shows the morphology of PT with homogenously distributed and

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interconnected pore structure. The pore size here was ranging from 10 µm to 400 µm, which could benefit the preservation for tissue volume, provide the temporary mechanical function, and also deliver biofactors [33]. The porosity of the sample was in the range of 60% to 70%, with open porosity from 70% to 80%. Fig. 1b shows the XRD patterns of the PT and MPT, respectively. For MPT sample, TiO peak appeared and Ti peak intensity obviously decreased, which indicated the bioactive TiO phase layer obtained on PT by MAO treatment. Fig. 1c shows the morphology of MPT. The oxide layer exhibited micro/nano porous structures and the pores were in a range of 50-100 nm. However, no detectable nano-porous structure was observed on PT in Fig. 1d. The results indicated that MAO treatment was successfully used to modify the surface of PT. The oxide film layer and micro/nano porous structures could be beneficial to bone-like apatite formation and exhibit an optimum bioactivity [34, 35].

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3.2 Gelatin microspheres and silver loaded microspheres The oil-in-water emulsion technique was used to fabricate gelatin microspheres

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which are easily controlled and collected. The average diameter of gelatin microspheres can be adjusted through the emulsification time and emulsifier dose.

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From the image of Fig. 2a, the average diameter of the microspheres was 4.46 µm, approximately, above 80% of samples in a range of 2-6 µm. Fig. 2b shows that the surfaces of microspheres were very smooth and no deposits accumulated on the

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microspheres. This reaction should relate to the Schiff-base reaction mechanism, which involve NH2 groups and ε-amine groups of lysine or hydroxylysine residues

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interacting with carboxyl groups. TEM image shows the network structure of gelatin microspheres in Fig. 2c, and this polymer network structure could make a great

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improvement for drug controlled delivery system. Several physical properties like

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rheology, swelling, and thermodynamics could be adjusted by chemical cross-linking in the gelatin network structure [36].

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As shown in Fig. 3a, when Ag+ reduction occurred, Ag/GMSs suspension changed slightly darker. Meanwhile the Ag+ preferred to block some of the -NH groups (primary silver binding sites) of gelatin microspheres [23, 37]. Ag particles can

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also form on amino groups using seed-mediated growth [38]. Here, gelatin microspheres could not only act as the sites of –NH groups binding Ag+ ions and also provide a high surface area for surface functionalization. With the reduction of silver-nitrate, the Ag particles formed at the sites of Ag+ ions attached on GMSs as shown in Fig. 3b. For the XPS analysis of Ag in Fig. 3c, the splitting of the 3d doublet is 6.0 eV, which indicates that Ag+ on the surface of GMSs was reduced to metal Ag0 forming Ag particles (Fig. 3b). Ag/GMSs were embedded and sectioned to determine whether Ag got into the microspheres. The SEM image in Fig. 3d, together with EDS analysis, suggested that some silver particles may also existed within the network structure of the microspheres. Furthermore, the Ag/GMSs section with a thickness of 80 nm was examined by TEM and HR-TEM. In Fig. 3e, several particles appeared in the profile of Ag/GMSs. The HR-TEM image of Ag/GMSs section in Fig. 3f shows

ACCEPTED MANUSCRIPT the interplanar spacing of 0.25 nm corresponding to the (100) crystal faces of the cubic Ag. And an interface was easily observed between Ag phase and amorphous GMS phase. Therefore, it can be confirmed that Ag0 particles produced inside GMSs

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within network structure besides on the surface of GMSs.

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3.3 Porous titanium scaffold with silver loaded gelatin microspheres. Through immersion, Ag/GMSs were distributed into PT and MPT respectively. Fig. 4a shows a large number of Ag/GMSs were tightly and homegeneously attached

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on the pore walls of the PT. As type A gelatin has an isoelectic point (iep) of pH 8-9, the prepared gelatin microsphere was positively charged at pH=7.4. Meanwhile, even

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if the reduction reaction completed, there was still Ag+ remained on the surface of gelatin microspheres. In contrast, after the treatment of micro-arc oxidation, the

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surface of the oxide layer of TiO on sample was negative charged (iep=5.5) at pH=7.4.

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In addition, the micro/nano porous structures of MPT increased the specific surface area and surface energy, which could also be favor for Ag/GMSs immobilization as

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shown in Fig. 4b. From Fig. 4a and Fig. 4b, the number of silver loaded gelatin microspheres in the MPT sample was higher than that in PT. That is, MPT was more

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benefit to immobilize silver loaded microspheres than PT.

3.4 Ag released in vitro. Release behavior of Ag was investigated in vitro, as shown in Fig. 5. Ag

exhibited a burst release both in Ag/GMSs/MPT and Ag/GMSs/PT, and followed with a prolonged release over an extended period. The total amount of Ag released from Ag/GMSs/MPT was higher than that from Ag/GMSs/PT sample, and after the 9th day, the Ag release rate for both samples slowed down. In other words, Ag/GMSs/MPT was superior to Ag/GMSs/PT in terms of higher Ag content delivery. This phenomenon supported that the MPT sample was able to carry more silver loaded gelatin microspheres than PT. On the other hand, Ag0 particles embedded inside GMSs probably resulted in a prolonged release of silver in both samples.

ACCEPTED MANUSCRIPT 3.5 Cell viability assay Too high concentration of silver released is considered to be cytotoxic, and could result in osteoblast dysfunction and apoptosis [29, 39]. The incubation time of cells

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was extended to 5 days to monitor the impact of continuously released silver ions on cells. As shown in Fig. 6, the gelatin microspheres loaded samples showed good

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biocompatibility and no cytotoxicity compared with the control (PT). The silver loaded samples suppressed cell viability to some extent due to silver release as Fig. 6 represents, however, the difference is statistically insignificant. Hence, the silver

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loaded gelatin microspheres have little influence on cell viability on samples. This phenomenon is also demonstrated by fluorescence microscopy images in Fig. 7. Fig. 6

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together with Fig. 5, on the first day, Ag/GMSs/MPT with a severe burst release phenomenon was more unfavorable to cell proliferation than Ag/GMSs/PT. But after

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3 days, Ag/GMSs/MPT was better than Ag/GMSs/PT in supporting osteoblast cell

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viability.

A highly porous structure may be valuable as a depot for bioactive constituents.

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As shown in Fig. 7, yellow arrows indicated that more cells preferred to aggregate on pore walls both in GMSs/MPT and GMSs/PT. MPT was more beneficial to promoting the adhesion and proliferation of cells, which might attribute to two factors: the TiO

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layer and micro/nano structures on the sample. The firmly adherent TiO layer is a bioactive surface and can stimulate cell adhesion, spreading and proliferation [35]. Micro/nano porous structures are beneficial to fluid transporting and nutrient conversion for cell growth, besides that their great surface area and high surface energy contribute to bioactivity. In this way, the aforementioned inhibitive effects on cells afforded by silver were compensated to a great extent by the TiO layer and micro/nano porous structures from micro-arc oxidation treatment.

3.6 Antibacterial behavior Bacterial concentration is characterized by absorbance values. Generally, silver loaded samples show great antimicrobial effects. Very low OD values in Fig. 8 indicated that the silver loaded samples were able to inhibit the growth of the bacteria

ACCEPTED MANUSCRIPT more significantly than other samples. Besides the releasing of Ag+ ions from Ag/GMSs, Ag0 particles on GMSs could also minimize bacteria adhesion, prevent biofilm of bacteria formation and kill them [31, 32]. As more Ag/GMSs attached onto

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the MPT sample, the amount of silver particles was relatively high, Ag/GMSs/MPT samples showed a higher antibacterial ability than Ag/GMSs/PT. Though the gelatin

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microsphere as a carrier played an important role in controllable release, the antibacterial effect of the samples slightly declined with the extension of time. One of the important strategies to mitigate implant infection is to render the

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implant surface with antibacterial property by impeding the formation of a biofilm. The release of Ag+ and Ag0 on Ag/GMSs prevented bacterial colonization and should

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inhibit the formation of biofilm on the material surface in the initial time. So, the antimicrobial effect of silver loaded samples was also confirmed by bacterial

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morphology as shown in Fig. 9. On the fifth day, the number of bacteria attached on

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the surface of Ag/GMSs/PT (a3, b3) and Ag/GMSs/MPT (a4, b4) was less than that of GMSs/PT (a1, b1) and GMSs/MPT (a2, b2). Yellow arrows showed different degrees

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of damages in the topography of bacteria, which was consistent with the results in Fig. 8. The number of damaged cells both E. coli and S. aureus bacteria was higher on Ag/GMSs/MPT sample than on Ag/GMSs/PT. On the other hand, the number of

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bacteria adhered on the surface of GMSs/MPT sample (a2, b2) was much more than that of GMSs/PT sample (a1, b1). This phenomenon should attribute to the bioactive TiO layer and micro/nano porous structures of samples after micro-arc oxidation treatment, which also benefited for the adhesion and proliferation of gram-negative and gram-positive bacteria.

4.Conclusions

The porous titanium (PT) and micro/nano porous titanium (MPT) with silver loaded microspheres serving as antibacterial implants were prepared. Ag0 particles attached on the gelatin microspheres and distributed inside the network structure. The gelatin microsphere vector can control the silver release rate. With the degradation of

ACCEPTED MANUSCRIPT GMSs gradually, both Ag+ ions and Ag0 particles make a contribution to antibacterial activity. The micro-arc oxidized samples with TiO layer and micro/nano porous are not only favorable to cells survival as compared to the others, but also support the

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attachment of the silver loaded gelatin microspheres.

In this study, both Ag/GMSs/PT and Ag/GMSs/MPT have great potential in

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antibacterial, and have an insignificant influence on cell viability. However, initial burst release is obvious, which may relate to a large number of Ag0 particles distributed on the surface of GMSs. Further investigations will focus on overcoming

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burst release by changing the degree of cross linking of gelatin in microspheres and increasing the amount of Ag0 particles inside the GMSs. In addition, other

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biodegradable polymers will also be in consideration to take place of gelatin. This work should be helpful to provide a novel approach of preparing bioactive bone

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Acknowledgement

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implants with high antibacterial ability.

This work was supported by the National Basic Research Program of China (973

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Program, 2012CB933600), Natural Science Foundation of China (31570955), Applied Basic Research Programs of Sichuan Province, China (2015JY0036) and Seedling Program of Science and Technology Department, Sichuan Province, China (2014-079).

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[39] J.M. Ahn, H.J. Eom, X. Yang, J.N. Meyer, J. Choi, Chemosphere, 108 (2014)

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Figure captions: Scheme1. Preparation scheme of silver loaded gelatin microspheres

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Fig. 1 SEM image of PT (a); XRD patterns of PT and MPT (b); SEM image of

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MPT (c); SEM image of PT (d).

Fig. 2 Size distribution graphs of gelatin microspheres (a); SEM image of cross-linked gelatin microspheres (b); TEM image of gelatin microspheres (c).

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Fig. 3 photographs of the samples (a), left hand tube contains GMSs and right hand tube contains Ag/GMSs; SEM image of Ag/GMSs (b); XPS spectrum of Ag3d

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region (c); SEM image and EDS spectum of Ag/GMSs section (d); TEM and HR-TEM images of Ag/GMSs section (e and f).

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Fig. 4 SEM images of the samples. (a) Ag/GMSs/PT, (b) Ag/GMSs/MPT. Fig. 5 Cumulative Ag release profiles of Ag/GMSs/PT and Ag/GMSs/MPT.

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Fig. 6 Time-dependent cell viabilities on the samples up to 5 days.

Fig. 7 Fluorescence microscopy images of the living cells on samples after 5 days of

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incubation displayed by DAPI fluorescence staining. (a) GMSs/PT, (b) GMSs/MPT, (c) Ag/GMSs/PT, (d) Ag/GMSs/MPT

Fig. 8 Absorbance values of four samples cultured after 1, 3 and 5 days with bacteria E. coli (a) and S. aureus (b), and blank control contains bacteria only; antibacterial efficiencies after 1, 3 and 5 days of Ag/GMSs/PT and Ag/GMSs/MPT samples against E. coli (c) and S. aureus (d).

Fig. 9 SEM images of E. coli cultured on the four samples (a1) GMSs/PT, (a2) GMSs/MPT, (a3) Ag/GMSs/PT and (a4) Ag/GMSs/MPT, and S. aureus cultured on the four samples (b1) GMSs/PT, (b2) GMSs/MPT, (b3) Ag/GMSs/PT and (b4)

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Ag/GMSs/MPT after five days.

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Scheme 1

ACCEPTED MANUSCRIPT Highlights:  Gelatin mirospheres (GMSs) were prepared as silver carrier, and Ag 0

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particles produced inside GMSs within network structure besides on

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the surface of GMSs.

 The oxide film layer and micro/nano structures not only benefits osteoblast attachment and proliferation, but also can provide more conditions

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 This work should be helpful to provide a novel approach of preparing

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bioactive bone implants with high antibacterial ability.