ZnO-Incorporated Hydroxyapatite Composite Coatings

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Nano Ag/ZnO Incorporated Hydroxyapatite Composite Coatings: Highly Effective Infection Prevention and Excellent Osteointegration Yanzhe Zhang, Xiangmei Liu, Zhaoyang Li, Shengli Zhu, Xubo Yuan, Zhenduo Cui, Xianjin Yang, Paul K Chu, and Shuilin Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17351 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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ACS Applied Materials & Interfaces

Nano Ag/ZnO Incorporated Hydroxyapatite Composite Coatings: Highly Effective Infection Prevention and Excellent Osteointegration

Yanzhe Zhanga, Xiangmei Liua, Zhaoyang Li b,Shengli Zhu b, Xubo Yuan b, Zhenduo Cuib, Xianjin Yangb, Paul K Chuc, Shuilin Wua,b* a

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials,

Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei Key Laboratory of Polymer Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China b

School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China

c

Department of Physics and Department of Materials Science and Engineering, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

* To whom correspondence should be addressed: E-mail: [email protected]; [email protected] (S.L. Wu)

Keywords: Antibacterial; Implant; Osteointegration; Laser cladding; Composite coating; Infection prevention

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Abstract The interfacial characteristics play an important role in infection prevention and osteointegration of artificial bone implants. In this work, both Ag and ZnO nanoparticles are incorporated into hydroxyapatite (HA) nano powders and deposited onto Ti6Al4V (Ti6) implants by laser cladding. The composite coatings possess a hierarchical surface structure with homogeneous distributions of Ag and ZnO. The Ag and ZnO nanoparticles are immobilized by laser cladding ensure long-term and gradual release of Ag and Zn ions at low cumulative concentrations of 36.2 and 56.4 µg/L after immersion for 21 days. A large concentration of Ag released initially increases the cytotoxicity but the large initial ZnO content enhances the cell viability and osteogenetic ability. The nano Ag/ZnO embedded HA coating (Ag: ZnO: HA= 7: 3: 90, wt%, namely Ag7ZnO3HA) exhibits optimal antibacterial efficacy and osteogenetic capability, as exemplified by the broad spectrum antibacterial efficacy of 96.5% and 85.8% against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively together with enhanced osteoinductivity with higher ALP activity of 134.60 U/g protein compared to 70.79 U/g protein for the untreated implants after culturing for 7 days. The rabbit femoral implant model further confirms that the optimized composite coating accelerates the formation of new bone tissues indicating 87.15% of newly formed bone area and osteointegration showing 83.75% bone-implant contact area even in the presence of injected S. aureus. The laser cladded Ag7ZnO3HA composite coatings are promising metallic implants with excellent intrinsic antibacterial activity and osteointegration ability.

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1. Introduction There is increasing demand for artificial orthopedic implants because of rapid development of orthomorphia, increasing incidents of fracture and joint degeneration, and bone defects due to the aging population.1 For example, in the United States, the market for total hip implants is about 572,000 annually and the demand for total knee arthroplasties is estimated to be 3.48 million by 2030.2 Because of the shortage of autograft bone and immunorejection of allograft bone, the development of artificial implant materials is important and necessary. Clinically, metallic biomaterials are commonly used in fixation of bone fracture and implantation of joint prostheses during the healing process because they possess good mechanical strength, low modulus of elasticity, and acceptable biocompatibility. In particular, Ti6Al4V (Ti6) has been extensively studied for orthopedic applications.3-4 However, many biometals are bioinert thus delaying the healing process and bacterial infection often occurs since they do not possess inherent antibacterial characteristics leading to implant failure. Clinically, implants-related bacterial infection is commonly treated by antibiotics but abuse of antibiotics can lead to bacterial resistance.5 In addition, the therapeutic effects of antibiotics are poor after bacterial biofilms form on the implants. To combat and resist bacterial infection, design of anti-adhesive surfaces, release of antimicrobial agents, antibacterial coatings, nanostructured materials, and molecules including antibiotics interfering with bacterial biofilms are viable strategies.6-13 In order to avoid the drug resistance, inorganic antimicrobial agents have been widely incorporated into coatings to kill bacteria due to the excellent electrical and optical properties.14-16 Among them, Ag nanoparticles (AgNPs) and ZnO NPs exhibit broadspectrum antimicrobial ability via release of the corresponding ions or production of 3



radical oxygen species (ROS) under light irradiation.8, 17-20 In addition, a combination of multiple inorganic NPs can produce better effects than a single component due to the synergistic effects.21-24 However, some side effects may be introduced by these methods. For example, release of inorganic NPs can cause toxicity14, 25 and a large concentration of Ag ions induces cytotoxicity.26 Furthermore, most methods cannot improve the osteogenetic ability of bioinert implants while maintaining the effective antibacterial activity. The poor bonding between implants and newly formed bone tissues can also lead to the loosening of implants and final failure27 and so it is urgent to develop new coatings with good osteoinductivity and antimicrobial ability at the same time. Hydroxyapatite (HA) with almost the same chemical composition as natural bone and tooth has excellent biocompatibility and osteoinductivity.28 In addition, nano-scale HA can bind fibronectin and vitronectin which are the key ligands of integrins playing important roles in cell adhesion.29-31 Besides, the antibacterial performance, ZnO shows excellent osteogenic properties by affecting bone metabolism. It can promote the expression of osteoblast marker gene and also stimulate mineralization of osteoblasts by depositing calcium on mesenchymal stem cells (MSCs).32-33 In view of this, the combination of HA, AgNPs, and ZnO NPs may produce the dual functions of intrinsic bacterial resistance and osteoinductivity. However, it is difficult to immobilize the three components on the surface of metallic implants simultaneously. Laser cladding can change the specific surface area, surface roughness, and wettability of implants to affect adhesion and differentiation of osteoblasts as well as Ca2+ absorption.34-37 Furthermore, the roughness and wettability of implants influence the bacterial resistance and inflammation38-39 but the most important issue for ceramic 4


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powders is how to fix them tightly on the surface of metallic implants.40 In this work, composite coatings composed of AgNPs, ZnO NPs, and HA nanopowders with different ratios are prepared on the Ti6Al4V alloy by laser cladding and the antibacterial efficacy and osteointegration are evaluated in vitro and in vivo tests. The process iss schematically illustrated in Scheme 1.

2. Experimental procedures

2.1 Materials preparation 2.1.1 Ti6Al4V pretreatment The Ti6Al4V (Ti6) (Shanghai Baosteel Co. Ltd., China) samples was cut into disks with dimensions of 6 mm × 2.5 mm. The Ti6Al4V plates were ground by SiC paper (240 grits), ultrasonically cleaned with ethanol and deionized water successively for 30 minutes, and dried in an oven at 37 oC for further use. 2.1.2 Preparation of mixed powders Five powder samples with different ratios of AgNPs, ZnO NPs, and HA nano powders were prepared (Table 1). The HA nanopowders (purity > 99.5%, BET, Aladdin) had an average particle size about 100 nm and ZnO NPs (purity > 99.8%, BET, Aladdin) were about 50 nm in size. The AgNPs were synthesized by a chemical reduction method. Briefly, the AgNO3 crystal particles were dissolved in 30% aqueous ammonia. The HA powders were dissolved in deionized (DI) water and sonicated for 30 min after adding the 5% dispersing agent of polyvinyl pyrrolidone (PVP). The HA solution was stirred for 1 h using a magnetic stirrer and then the silver ammonia solution was added to the HA solution. The aqueous hydrazine hydrate (N2H4) solution was added dropwise to reduce form metallic silver. During the 5



reduction reaction, the white solution gradually changed to grey and after the solution became grey, the nano Ag/HA powders were filtered, rinsed with alcohol and DI water several times, and dried in an oven at 60 oC. Finally, the Ag/HA mixed with ZnO nanoparticles were ground by a quartz mortar. Composites with different amounts of silver were prepared by adjusting the AgNO3 solution and different ZnO contents were determined by weighing. The Ag/ZnO/HA (AgxZnOyHA) composite powders were prepared in a 10 mg/mL suspension in DI water with proportions shown in Table 1. 100 µL of the AgxZnOyHA suspension was added to the Ti6 substrate and dried at 60 oC in a vacuum oven prior to cladding. 2.1.3 Laser cladding Laser cladding was performed using a CW 2 kW Nd:YAG laser (wavelength 1.06 µm, LUMONICS, JHM-1GY-300B) transmitted via an optical fiber and focused onto the samples. To prevent oxidation of AgNPs, high-purity argon gas was applied to the interaction zone. The quality of the layer depends on processing parameters such as the laser current (I), laser pulse width (W), laser frequency (f), laser spot diameter (D), and scanning speed (V). The coating could not be completely melted at a low laser fluence resulting in loose bonding between the coating and substrate. On the contrary, a high laser fluence leads to excessive melting between the substrate and coating, and silver and zinc ions cannot be released easily from the coating. Based on experimental trials, the optimal parameters were I = 100 A, W = 2 ms, f =20 Hz, D = 0.6 mm, and V = 5 mm/s.

2.2 Materials characterization The microstructures and composition of the composite coatings were characterized by transmission electron microscopy (TEM; Tecnai G20, FEI, USA), 6


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selected-area electron diffraction (SAED), energy dispersive X-ray spectrometry (EDS). The surface morphology was examined by field-emission scanning electron microscopy (FESEM, JSM7100F) and the chemical states were determined by X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific 250Xi) with Al Kα radiation (1486.6 eV).

2.3 Zn2+ and Ag+ release The samples were immersed in 30 mL of simulated body fluid (SBF) at 37 oC for 1, 4, 7, 14 and 21 days, respectively and the solution was collected and refilled with 3 mL of fresh SBF at pre-set intervals. The Zn2+ and Ag+ concentrations in the solution were determined by atomic absorption spectrophotometry (AAS W006, analytikjena, Germany).

2.4 Contact angle measurement The contact angles were determined at room temperature on a contact angle instrument (OCA40 Filderstadt, Germany) with DI water as the medium. The average calculated from 3 drops at different positions was calculated.

2.5 Antibacterial activity 2.5.1 Spread plate To investigate the antibacterial activity of the laser cladding AgxZnOyHA coatings, Gram-negative Escherichia coli (E. coli, ATCC 8099 ) and Gram-positive Staphylococcus aureus (S. aureus, ATCC 25923) of standard strains were used. The bacteria were cultured in the Luria-Bertani (LB) culture medium and LB broth and the LB agar plates were sterilized by autoclaving at 121 oC for 15 min. Three samples in 7



each group were tested to obtain averages. The samples were placed on a 48-well plate and 400 µL of the diluted E. coli suspension (1 × 107 CFU/mL) in the sterile PBS broth were in a biological safety cabinet and incubated at 37 oC for 12 h. Afterwards, the E. coli suspension in each hole was diluted 100 folds by sterile PBS and 20 µL of the diluted E. coli suspension was drawn from each hole to spread on LB agar plates and incubated in the oven at 37 oC for 24 hours. The same experimental protocol was adopted for S. aureus. The antibacterial efficiency of the samples was calculated by the following formula according to the number of colonies on the plate:

Antibacterial efficiency(%)=

Control numbers - Experiment numbers × 100% (1) Control numbers

2.5.2 Bacterial morphology The bacteria on the samples were fixed with 2.5% glutaraldehyde for 2 h, dehydrated sequentially in a series of ethanol with different concentrations (30, 50, 70, 90, and 100 v/v %) for 15 min each, and freeze-dried. The morphology of the bacteria was observed by SEM after gold plating.

2.6 Cell culture The mouse calvarial cell line MC3T3-E1 was used in this study. The cells were usually cultured on a dish in the medium composed of α-MEM (HyClone) supplemented with 1% penicillin−streptomycin (HyClone) and 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 at 37 oC in an incubator. The cells were treated with sterilized PBS and trpsin/EDTA (Sigma) to remove dead ones and then diluted to a density of 1 × 105 cells/mL for further use. 2.6.1 Cell viability and proliferation 8


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The cell viability was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyl tetrazolium bromide (MTT) assay. The sterilized samples were put on 96well culture plates in 150 µL of the medium with a density of 1 × 105 cells/mL in each well and incubated for 1, 3, and 7 days, respectively. After removing the medium from each well, 100 µL of the MTT solution (0.5 mg/mL) were added and incubated for 4 h at 37 oC until the ianthinus precipitate was visible. The MTT solution was removed and 100 µL of dimethyl sulfoxide (DMSO) were added to each well to dissolve the ianthinus precipitate. Finally, the samples were taken out and the optical density at 490 nm was determined on a microplate reader (SpectraMax I3, Molecular Devices). The cell viability was calculated by the ratio of the experimental group and control group. The culture medium was refreshed every 3 days and experiments were done on three parallel groups. 2.6.2 Osteogenic differentiation The osteogenic activity was observed by the ALP activity assays according to Microplate test kit of manufacturer’s instruction (ALP assay kit). The samples were put on 96-well culture plates and 150 µL of the medium with a density of 1 × 105 cells/mL were added to each well and incubated for 3, 7, and 14 days, respectively. After incubation, the medium was sucked out from each well and 1% Triton X-100 was added. The ALP activity was measured at 520 nm on a microplate reader. Besides, the protein content of the cell in each well was tested using BCA protein assay kit (Solarbio) at 562 nm on a microplate reader, which can get the ALP activity per unit protein. The culture medium was refreshed every 3 days and triplicate experiments were performed. 2.6.3 Osteogenic mineralization Four groups including the sterilized Ti6, HA-Ti6, and Ag7ZnO3HA-Ti6 samples 9



were placed on four 96-well plates with 150 µL of the medium with a density of 1 × 102 cells/mL in each well. The medium was refreshed every 3 days. After culturing for 10 days, one group was examined by SEM and EDS to observe the cell morphology and determine the elements. Another group after alizarin red staining was observed for mineralized nodules. Calculation was performed after culturing for 20 days and the same protocol was implemented on the other two groups. Three parallel experiments were conducted for each group.

2.7 In vivo animal tests 2.7.1 Surgical protocol The in vivo animal experiments were approved by Wuhan Service Biotechnology Co., Ltd., China. Adult male New Zealand white rabbits weighing about 2.5 kg were obtained from Wuhan Center for Disease Prevention & Control and 12 rabbits were randomly divided into two groups, i.e., 6 rabbits in the control group (Ti6) and the other in the experimental group (Ag7ZnO3HA-Ti6). Before implanting into the rabbit femur, 3% pentobarbital sodium salt was used as an anesthetic to treat the rabbit (1mL/kg). The hair on the right leg femur was shaved and disinfected with 1% povidone iodine and sterilized surgical instruments were used to perform surgery along the direction of the femoral axis. The skin and muscle of the rabbit femur were cut until the femur was exposed. The implants were seeded with 20 µL of S. aureus (105 CFU/mL) and a suitable gap was sawed from the exposed femur. The sample was implanted into the femur and placed into the bone marrow cavity (Fig. S7a) and the incision was stitched. The other rabbits underwent the same surgical protocol and all the rabbits were housed in individual sterilized cages. 2.7.2 Characterization 10


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After two weeks, three rabbits from each group were sacrificed by injection of excessive anesthetics. The soft and bone tissues in contact with the samples were extracted to evaluate bacterial infection by hematoxylin and eosin (H&E) staining and Giemsa staining. At 5 weeks post-surgery, the remaining rabbits were sacrificed to assess tissue inflammation and osteogenesis. These evaluation methods included Xray photography, micro-CT analysis, H&E staining, Giemsa staining, and Van Gieson's picro fuchsin staining. 2.7.3 X-ray and Micro-CT assessment At 5 weeks post-surgery, X-ray pictures were taken from the sacrificed rabbits to observe bone growth. High-resolution micro-computed tomography (Micro-CT; SkyScan 1176) was conducted at 60 kV and 400 µA. A 1 mm aluminum filter was used to determine the newly formed bone around the implants and the 2D and 3D osteogenic models were constructed using the micro-CT scanning software. 2.7.4 Histo-pathological evaluation H&E staining and Giemsa staining were used in the histological analysis to evaluate the inflammatory response of soft tissues and bones around the implants. At 2 and 5 weeks after operation, the soft tissues and bones were fixed in 4% paraformaldehyde for 24 h and the bone tissues were additionally treated with decalcification for a month. The samples were dehydrated for 1 h in each stage with graded alcohol (75, 85, 90, 95, and 100%) and treated with xylene. The tissues were embedded into paraffin and sectioned using a microtome (Leica RM2016, LeicaMicrosystems, Germany).

The histological sections were divided into two

groups for H&E staining and Giemsa staining and the inflammatory response and bacterial infection were examined by optical microscopy. New bone formation around the implants was evaluated by Van Gieson's picro 11



fuchsin staining. At 5 weeks after operation, the implants and surrounding bone tisues were cut off together and fixed in 4% paraformaldehyde for 3 days. The hard tissues were then dehydrated with graded alcohol (70, 90, 95, and 100%), respectively, and treated with xylene for 3 days. After sealing with bymethyl methacrylate (MMA), the embedded hard tissues were sliced using a microtome and polished to about 10 µm. The histological sections were stained with Van Gieson's picro fuchsin. The new bones were dyed red and observed under an optical microscope. The newly formed bone rates and contact rate between the bone and implants were determined by ImageJ.

2.8 Statistic analysis The results were calculated as mean ± standard deviation (SDs) based on at least three experiments according to Kruskal-Wall in the one-way analysis of variance (ANOVA).

3. Results and discussion

3.1 Surface characterization The mixed powders of AgxZnOyHA are characterized by TEM. As shown in Fig. S1a, the commercial HA nanopowders are composed of many needle-like nanorods. The SAED pattern discloses that these nanorods have different orientations (Fig. S1b). The mixed powders with reduced AgNPs exhibit similar structures (Fig. S1c) and SAED patterns (Fig. S1d) due to the small amount of AgNPs from the AgNO3 solution. The size of the AgNPs ranges from 30 to 60 nm. As shown in Fig. S1e, O, Ag, Ca, and P are detected by EDS demonstrating the co-existence of HA and 12


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Ag in the mixed powders. The copper signal originates from the copper mesh. The surface morphology of the AgxZnOyHA composite coating on Ti6 in Fig. 1a shows a regular texture with parallel ridges because rapid melting and freezing occur as the laser spot moves back and forth quickly on the surface. There are bulging areas (zone 1) and flat areas (zone 2). The high-magnification image of the bulging areas (Fig. 1b) reveals raised molten peaks while that of flat areas shows a loccular nanostructure (Fig. 1c). The cross-sectional SEM image of AgxZnOyHA-Ti6 shows good bonding between the coating and substrate and the largest cladding depth is about 460 µm as indicated by red arrows (Fig. 1d). In the Ag5ZnO5HA-Ti6 sample, the EDS scan discloses homogeneous distributions of Ca, P, Ti, Zn, and Ag in the coating (Fig. 1e) confirmed by the XPS survey scan (Fig. 2a). As shown in Fig. 2b, the XPS narrow scan discloses Ag 3d5/2and Ag 3d3/2 peaks at 368.0 and 374.0 eV, respectively, and the difference between Ag 3d5/2and Ag 3d3/2 is 6.0 indicative of the metallic state in the coating.41 As shown in Fig. S2, the contact angle on the untreated Ti6 is 73.7o, whereas that on the laser treated Ti6 (L-Ti6) decreases to 24o. After cladding of HA, the contact angles on the AgxZnOyHA decrease further with the HA cladded sample showing the smallest contact angle of 10.5o. On the flat surfaces, the wettability depends on the surface free energy that can be expressed by Young's equation:

γsv =γsl +γlv cosθY , (2) where θY is the contact angle, γ refers to the surface tension (or surface free energy), and s, l, and v represent the solid, liquid, and gas, respectively. For the droplet on the untreated Ti6, the relationship between the contact angle and surface tension is expressed by equation (2), but for the uneven surfaces, the surface roughness plays an important role in the wettability. The relationship between the roughness and contact 13



angle is expressed by Wenzel equation:

cosθW = r cosθY , (3) where r represents the surface roughness factor, θw is the contact angle in the Wenzel model, and θY is the contact angle in Young’s model. The roughness affects the

wettability when the contact angle of the flat surface is less than 90o.42-43 Compared to the untreated Ti6, the contact angle on L-Ti6 decreases due to the rough surface after the laser treatment during which there is heat transfer from the HA and AgxZnOyHA composite coatings to the Ti6Al4V substrate. The laser beam size is 0.6 mm and the cooling rates at the center and periphery are slightly different forming nano-scale microspheres during solidification. The molten pool structure forms at a laser frequency of 20 Hz and the low laser energy produces the bulging areas in the coating between pulses. The parallel ridges and nano-scale molten pools form a hierarchy structure (shown in Fig. 1) further increasing the hydrophilicity of the HA and AgxZnOyHA samples. The OH group in HA increases the hydrophilicity and so the HA-cladded Ti displays the smallest contact angle. It is believed that a hydrophilic surface favors adhesion of osteoblasts fostering subsequent growth and proliferation.5

3.2 Release behavior of Zn2+ and Ag+ Zn2+ and Ag+ are linked to the antibacterial activity of coatings.8 Zn is an essential trace element playing an important role in regulation of osteoblast functions and bone formation.32 The release behavior of Zn2+ in the SBF solution is shown in Figure 3a.

In the earlier stage within four days of immersion in SBF, all the

AgxZnOyHA-Ti6 samples exhibit burst release of Zn2+ due to the reaction between exposed ZnO and electrolyte at a pH of 7.42 as follows: ZnO + H 2O ↔ Zn 2 + + 2OH − (4) 14


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As the ZnO content in the composite coatings increases, the amount of released Zn2+ increases as shown in equation (4). The cumulative concentration of Zn2+ released from Ag0ZnO10HA-Ti6 is 46.2 µg/L after immersion for 4 days and the release rate diminishes gradually afterwards.

After immersion for 14 days, the cumulative

concentration of Zn2+ reaches a stable value for all samples, i.e., 20.11, 28.74, 36.49, and 54.5 µg/L for Ag7ZnO3HA-Ti6, Ag5ZnO5HA-Ti6, Ag3ZnO7HA-Ti6, and Ag0ZnO10HA-Ti6, respectively. The corresponding increments are 1.71, 1.70, 3.13, and 1.91 µg/L after immersion for 21 days indicating chemical stability. It is noted that the largest cumulative Zn2+ concentration (56.41 µg/L from Ag0ZnO10HA-Ti6) is far below the safety level of 3 mg/L recommended by the World Health Organization.44 The release profiles of Ag+ from the AgxZnOyHA-Ti6 samples are presented in Fig. 3b. Compared with Zn2+, there were no obvious burst release of Ag+ and all the samples exhibit slow and sustained release of Ag+ at lower concentrations because release of Ag+ from nano-silver in the composite coatings arises from ionization of the outer Ag atoms of the nano-silver in PBS. As the Ag content increases, the cumulative amount of Ag+ released increases. The cumulative concentration of Ag+ released from Ag10ZnO0HA-Ti6 increase to 33.59 µg/mL after immersion for 14 days and afterwards, the released amount increases gradually to 37.68 µg/mL after 21 days. With regard to Ag containing antibacterial composites, previous studies have shown larger Ag release.45-46 The doping process by physical mixing of HA or brushite cements in the AgNO3 solution leads to rapid release of Ag+ at a higher concentration of over 200 µg/L after immersion in PBS for 7 days,45 whereas that released from the HA/Ag composite coating prepared by plasma spraying is larger than 300 µg/L after immersion for 14 days,46 indicating that Ag is immobilized in the composite coatings. A larger Ag concentration kills bacteria more 15



effectively but comes with toxicity.47 In addition, rapid release of Ag+ does not bode well for the long-term antibacterial activity. Here, laser cladding can fix the composite coating tightly on the Ti6 substrate due to the high transient temperature consequently blocking rapid release of Zn2+ and Ag+ from the AgxZnOyHA composite coatings and reducing the associated toxicity. Slow and gradual release of Ag+ is preferred for long-term prevention of bacterial infection. Although the melting point of nano-silver is size-dependent, it is similar to that of bulk Ag if the particle size is over 10 nm.48 Hence, owing to the lower melting point (961.78 ℃) and higher thermal conductivity (473 W·m−1·k−1),49-50 tight fixation of Ag is accomplished.

3.3 In vitro antibacterial activity of AgxZnOyHA-Ti6 The antibacterial activity of the AgxZnOyHA composite coatings is evaluated by the plate counting method. As shown in Fig. S3a, with the exception of L-Ti6, the other samples exhibit different levels of antibacterial efficacy against E. coli relative to pure Ti6 as determined by the specific bacterial count on the LB agar plates (indicated with red number) ascribed to the antibacterial action of released Ag+ and Zn2+ or the synergistic effects of both ions. L-Ti6 shows more E. coli than the untreated sample due to the hierarchical structure produced by the laser treatment (Fig. S4), which enhances the hydrophilicity (shown in Fig. S2) and favors the adhesion of bacteria. HA-Ti6 exhibits slight reduction in the bacterial strains than the untreated sample due to the following two factors. On the one hand, the surface hydrophilicity for a contact angle of 10.5o favors adhesion of bacteria but on the other hand, HA nanoparticles have some antibacterial activity.51

The E. coli numbers decrease

significantly with contents of AgNPs in the AgxZnOyHA coatings. According to formula (1) and bacterial strain numbers in Fig. S3a, the antibacterial efficacy of samples against E. coli is calculated (Fig. 4a). The antibacterial efficiency of L-Ti6 16


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was -9.4% but 5.34% for HA-Ti6.

Addition of ZnO (10 wt%) improved the

antibacterial efficacy by up to 53.3% against E. coli due to released Zn2+. As the Ag content is increased to 3 wt%, the antibacterial efficacy against E. coli increases sharply to 79.6% and the antibacterial rates against E. coli are 90.4%, 96.5%, and 99.2% for Ag5ZnO5HA-Ti6, Ag7ZnO3HA-Ti6, and Ag10ZnO0HA-Ti6, respectively. Fig. S3b shows the plate counting results of S. aureus showing a similar trend. That is, L-Ti6 has more S. aureus than the untreated Ti6 but HA-Ti6 has less bacteria than the control. A hydrophilic and nanostructured surface has been shown to favor adhesion of bacteria.52-53 The antibacterial rates of L-Ti6, HA-Ti6, Ag0ZnO10HATi6, Ag3ZnO7HA-Ti6, Ag5ZnO5HA-Ti6, Ag7ZnO3HA-Ti6, and Ag10ZnO0HA-Ti6 against S. aureus are -21.9%, 6.5%, 35.9%, 54.3%, 77.1%, 85.8%, and 93.0%, respectively (Fig. 4b). Addition of ZnO and Ag nanoparticles endow the coatings with antibacterial ability. Addition of ZnO (10 wt%) inhibits the growth of S. aureus by about 35.9% without the assistance of AgNPs whereas addition of Ag (10 wt%) only enhances antibacterial efficacy to 93%, indicating the predominant role of AgNPs in bacteria killing. Fig. 4c shows the morphology of the bacteria on different samples. Both E. coli and S. aureus show an irregular smooth shape with intact cell walls but on Ag0ZnO10HA-Ti6, Ag7ZnO3HA-Ti6, and Ag10ZnO0HA-Ti6, the cell walls show different degrees of damage such as sunken, cracked and deformed features (indicated by red arrows). The damage becomes more serious as the AgNPs content is larger. It has been reported that AgNPs and Ag+ released from AgNPs leads to intracellular oxidation and membrane potential variation resulting in destruction of bacteria proteins54-55 and AgNPs act as a vehicle to deliver Ag+ to generate ROS to destroy the bacteria DNA.26, 56

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3.4 In vitro evaluation of cell viability and mineralization The Cell viability evaluated by MTT on days 1, 3, and 7 is shown in Fig 5a. After culturing for 1 day, except Ag10ZnO0HA-Ti6, all the samples exhibit better cell viability than the pristine Ti6, due to the more amount of Ag+ released from the Ag10ZnO0HA-Ti6 sample than other samples (Fig. 2). The better cell viability of LTi6 is ascribed to the hierarchical nanostructure.

Among the AgxZnOyHA-Ti6

samples, HA-Ti6 shows the largest cell survival rate because HA has a similar composition as natural bone thus facilitating osteoblast proliferation. However, after culturing for 3 days, except for HA-Ti6 and L-Ti6, the modified samples show reduced cell viability due to the higher cumulative concentrations of released Zn2+ and Ag+ as shown in Figs. 2a and 2b. Ag10ZnO0HA-Ti6 shows the lowest cell viability because of the largest concentration of Ag+. Compared to Ag nanoparticles, ZnO nanoparticles shows lower cytotoxicity.57 It has been reported that AgNPs induce oxidative stress58 impacting cell proliferation.56 On the 7th day, the cell viability of all the samples exhibited a similar trend as that on the 3rd day. Furthermore, except Ag10ZnO0HA-Ti6, the four composite coatings display increased cell viability on the 7th day compared to that on the 3rd day, indicating decreasing cytotoxicity with culture time. The continuous decrease in the cell viability observed from Ag10ZnO0HA-Ti6 provides evidence that more Ag+ release produces cytotoxicity. The ALP activity is an indicator of osteoblast differentiation and osteogenesis. As shown in Fig. 5b, on the 3rd day, the ALP activity of the untreated sample is 39.5 U/g protein and 42.1 U/g protein for L-Ti6. All the AgxZnOyHA-Ti6 samples show higher ALP expression than the untreated Ti6. After culturing for 7 days, the cells on AgxZnOyHA-Ti6 samples show enhanced ALP expressions indicating excellent osteogenesis properties. All the AgxZnOyHA composite coatings have higher ALP 18


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activities than the pure HA coating due to release of small amounts of Ag and Zn. On the 14th day, the ALP activity is similar to that after 7th day. The slightly higher ALP activity of L-Ti6 than the untreated sample is attributed to the larger roughness and hydrophilicity.59-60

Moreover, Ca and P from the HA provide the osteogenetic

mineralization factors.30, 61 The released Zn can be easily absorbed to promote bone proliferation and osteogenic mineralization by stimulating osteoblast metabolism. Zn also promotes deposition of calcium in HA33 and it has been reported that the local micro-galvanic couples formed between AgNPs and the Ti substrate favor bone formation.51, 62 The morphology of the MC3T3-E1 cells is examined by SEM and fluorescence staining. As shown in Fig. 5c, after culturing for 1 day, the MC3T3-E1 cells attach well and grow on all the samples. The MC3T3-E1 cells on the untreated Ti6 show symmetrical adhesion with smooth edges (indicated by the red arrow) but there are no obvious filamentous pseudopods, whereas those on L-Ti6 show short pseudopods (indicated by the red arrow). Compared to the untreated Ti6, larger spindles and long pseudopods are observed from the HA and AgxZnOyHA coatings (indicated by the red arrow), especially the AgxZnOyHA coatings. The fluorescence images (Fig. 5d) show the normal morphology. The numbers of cells on the AgxZnOyHA-Ti6 samples decreases gradually with Ag concentration in agreement with SEM and MTT after culturing for 1 day. Considering the effective in vitro antibacterial activity and excellent cytocompatibility of Ag7ZnO3HA, this group is selected for the mineralization and in vivo tests. Mineralization of the MC3T3-E1 cells can be evaluated by examining the formation of mineralization nodules by SEM and EDS. As shown in Fig. 6, after culturing for 10 days, the osteoblasts exhibit a smooth morphology on the untreated 19



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Ti6 and EDS shows intense signal of Ti from the substrate, indicating that the MC3T3-E1 cells do not undergo mineralization. Some dot-like protrusions appear on HA-Ti6 ls (indicated by blue arrows) and the EDS results obtained from a nodule show a more intense Ca peak than Ti, suggesting partial mineralization of the MC3T3-E1 cells. The mineralized nodules of the MC3T3-E1 cells on Ag7ZnO3HATi6 are larger than those on HA-Ti. In addition, the Ca signal is stronger than that from HA-Ti6 indicating better minerization on Ag7ZnO3HA. The Ca concentration in the mineralized nodule of MC3T3-E1 is larger than that in the non-mineralized areas (Fig. S5a). After culturing for 20 days, the MC3T3-E1 cells spread well and adhere tightly to the untreated Ti6. The corresponding EDS data show almost no Ca signal but an intense Ti peak indicative of no minerization.

Many mineralized

nodules appear on HA-Ti6 and EDS shows strong Ca peak but weak Ti peak compared to the sample cultured for 10 days indicating further minerization. The MC3T3-E1 cells on Ag7ZnO3HA-Ti6 exhibit a higher minerization level than those on HA-Ti6 as shown by the larger minerization nodules, intense Ca signal, and weak Ti signal.

The EDS spectrum obtained from the non-mineralized zone shows a

stronger Ti peak than that from the mineralized nodules (Fig. S5b). The alizarin red stained images of different samples are shown in Fig. S6a and no mineralized nodules appear on the untreated Ti6 after culturing for 10 days, whereas both HA-Ti6 and Ag7ZnO3HA-Ti6 display obvious red mineralized nodules. After culturing for 20 days, the untreated sample still does not show obvious stained nodules but HA-Ti6 and Ag7ZnO3HA-Ti6 exhibit more obvious alizarine red stained nodules, especially the latter one. The quantitative measurement of mineralization by alizarin red staining is shown in Fig. S6b. After culturing for 10 and 20 days, the mineralization level on Ag7ZnO3HA-Ti6 is the best (Fig. 6).

The excellent in vitro osteogenetic

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differentiation ability of Ag7ZnO3HA can be attributed to two factors. First of all, HA with the composition of natural bone benefits adhesion and proliferation of osteoblasts and Ca and P in HA are conducive to cell differentiation and mineralization. Secondly, released Ag and Zn released from Ag7ZnO3HA stimulate the metabolism and osteogenesis of osteoblasts as well as subsequent bone mineralization.29-30

3.5 In vivo antibacterial activity The in vivo antibacterial efficiency is assessed by the inflammatory response of soft tissues and bone tissues around the implanted model in vivo. H&E staining is a common immunoassay to evaluate inflammatory cells in tissues and Giemsa staining is employed to detect cell's eosinophil and bacteria. As shown in Fig. 7, after the samples have been implanted for 2 weeks, the soft tissues and bone tissues in the untreated Ti6 group show substantial inflammatory cell infiltration by H&E staining. The inflammatory cells are mainly lobulated neutrophils (indicated by red arrows) and as an inflammatory response in the immune system, neutrophils quickly attack the bacteria.63-64 Many bacteria are observed from the soft tissues and bone tissues by Giemsa staining (indicated by black arrows). In contrast, on the Ag7ZnO3HA-Ti6 implant, only a small number of lobulated neutrophils is distributed in the soft tissues and bone tissues as shown by the H&E staining images. Giemsa staining shows few bacteria in the soft tissues and bone tissues as well. After 5 weeks, H&E staining shows a lot of inflammatory cell infiltration including neutrophils in the soft and bone tissues of the untreated Ti6 group and many bacteria are shown in the Giemsa staining image. In comparison, the soft tissues and bone tissues around the Ag7ZnO3HA-Ti6 implant show almost no inflammatory cells and bacteria in the H&E and Giemsa 21



staining image. These results disclose that the Ag7ZnO3HA coating has excellent in vivo antibacterial efficacy in agreement with the in vitro antibacterial results. Ag and ZnO play synergistic roles favoring long-term anti-bacterial activity.

3.6 Bone-implant formation in vivo To evaluate the in vivo bone formation ability of Ag7ZnO3HA-Ti6 during bacterial infection, the rabbits injected with 20 µL of S. aureus at 105 CFU/mL are sacrificed after 5 weeks and inspected by X-ray radiography. As shown in Figs. S7a and S7b, the implant is tightly fixed to the surrounding tissues in the rabbit femur and newly formed bone tissues can be detected around the implant by micro-CT. The 2D and 3D models reconstructed from the CT images show the formation of new bone tissues around the implant. In the 2D model (Fig. 8a), it is difficult to find new bone tissues around the untreated Ti6 implant but the two sides of Ag7ZnO3HA-Ti6 are completely covered by newly formed bone tissues (indicated by red arrows). In the 3D model, the implants and newly formed bone tissues are marked red and yellow, respectively, and more bone tissues are observed from Ag7ZnO3HA-Ti6 than the untreated Ti6 implant. The osteogenesis ability can be evaluated by analyzing the ratio of Object Volume (Obj.V) and Tissue Volume (TV). The Obj.V/ TV value of the Ag7ZnO3HA-Ti6 implant is 46.01% but only 20.10% for the untreated Ti6 implant as shown in Fig. 8b. New bone formation around the implants and bone-implant contact is evaluated by Van Gieson's picro fuchsin staining. As shown in Fig. 8c, more new bone tissues (red color) form around Ag7ZnO3HA-Ti6 than the untreated Ti6 implant. The highmagnification images of the interface between the implant and surrounding tissues disclose larger bone-implant areas on Ag7ZnO3HA-Ti6 than the untreated Ti6. The 22


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new bone area coverage of the former is 87.15% and that of the latter is 44.36%. The contact area between bone tissues and Ag7ZnO3HA-Ti6 is 83.8% and so new bone tissues almost completely cover the implant. In contrast, most of the surface on the untreated Ti6 implant is covered by soft tissues (indicated by blue arrows). In the early recovery stage after surgery, the composite coating possesses the hierarchical nanostructure which benefits adhesion of osteoblasts and osteointegration65-66 and Ca and P facilitate formation of new bone tissues by promoting osteoblast mineralization. Besides killing bacteria, Zn and Ag promote bone cell metabolism and absorption of Ca and P33, 62 to accelerate bone formation.

4. Conclusion Composite coatings composed of AgNPs, ZnO, and HA nano powders are fabricated on metallic Ti6 implants by laser cladding which provides good adhesion between the coatings and substrate due to local melting during the laser treatment. Long-term release of both Ag+ and Zn2+ endows the implants with both effective bacterial resistance and osteogenetic ability. Laser cladding suppresses the release rate of Ag ions to mitigate the cytotoxicity at large silver concentrations and HA in the composite coatings enhances osteogenesis by accelerating mineralization of osteoblasts. Addition of ZnO reduces the initial content of AgNPs to weaken the cytotoxicity of Ag+ without impairing the antibacterial efficacy via the synergistic antibacterial effects of both Ag+ and Zn2+. The laser cladded AgxZnOyHA composite coatings have large potential providing intrinsic bacterial resistance, excellent osteogenesis, as well as rapid osteointegration.

Acknowledgements 23



This work is jointly supported by the National Natural Science Foundation of China, Nos. 51422102, and 81271715, the National Key Research and Development Program of China No. 2016YFC1100600 (sub-project 2016YFC1100604), Hong Kong Research Grants Council (RGC) General Research Funds (RGC) General Research Funds (GRF) No. CityU 11301215, and City University of Hong Kong Applied Research Grant (ARG) No. 9667144.

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56.Park, M. V.; Neigh, A. M.; Vermeulen, J. P.; de la Fonteyne, L. J.; Verharen, H. W.; Briede, J. J.; van Loveren, H.; de Jong, W. H. The Effect of Particle Size on the Cytotoxicity, Inflammation, Developmental Toxicity and Genotoxicity of Silver Nanoparticles. Biomaterials 2011, 32, 9810-9817. 57.Puzyn, T.; Rasulev, B.; Gajewicz, A.; Hu, X.; Dasari, T. P.; Michalkova, A.; Hwang, H. M.; Toropov, A.; Leszczynska, D.; Leszczynski, J. Using Nano-QSAR to Predict the Cytotoxicity of Metal Oxide Nanoparticles. Nat. Nanotechnol. 2011, 6, 175-178. 58.Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M. Toxicity of Nanomaterials. Chem. Soc. rev. 2012, 41, 2323-2343. 59.Thian, E. S.; Ahmad, Z.; Huang, J.; Edirisinghe, M. J.; Jayasinghe, S. N.; Ireland, D. C.; Brooks, R. A.; Rushton, N.; Bonfield, W.; Best, S. M. The Role of Surface Wettability and Surface Charge of Electrosprayed Nanoapatites on the Behaviour of Osteoblasts. Acta Biomater. 2010, 6, 750-755. 60.Gittens, R. A.; Olivares-Navarrete, R.; Cheng, A.; Anderson, D. M.; McLachlan, T.; Stephan, I.; Geis-Gerstorfer, J.; Sandhage, K. H.; Fedorov, A. G.; Rupp, F.; Boyan, B. D.;

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Figure captions Fig. 1 (a) SEM image showing the surface morphology of the Ag5ZnO5HA-Ti6 (5:5:90, wt%) composite coating; (b) SEM image of bulging area (zone 1); (c) SEM image of the flat area (zone 2); (d) Cross-sectional SEM micrograph; (e) Elemental distribution by EDS. Fig. 2 (a) XPS survey spectrum of Ag5ZnO5HA-Ti6 and (b) XPS spectrum of Ag of Ag5ZnO5HA-Ti6 sample. Fig. 3 (a) Release of Zn from different ZnO NPs and (b) Releasing of Ag from different AgNPs. Fig. 4 Antibacterial properties against the E. coli and S. aureus: (a) Antibacterial efficiency of the samples compared to Ti6 for E.coli; (b) Antibacterial efficiency of the samples for S. aureus; (c) SEM images of E. coli and S. aureus on Ti6, Ag0ZnO10HA-Ti6, Ag7ZnO3HA-Ti6, and Ag10ZnO0HA-Ti6 (scale bar = 1 µm). The error bars indicate means ± standard deviations (*P < 0.05, **P < 0.01 and ***P < 0.001 (t test)). Fig. 5 In vitro tests: (a) Cell viability assessed by MTT after culturing for 1, 3, and 7 days; (b) Cell osteogenesis evluated by the ALP activity assay for 3, 7, and 14 days; (c) SEM images and (d) Fluorescent images of the cells on the different samples after incubation for 1 day (Means ± SD, n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001 (t test)). Fig. 6 SEM images showing formation of bone minerals for MC3T3-E1 cells incubated on Ti6, HA-Ti6, and Ag7ZnO3HA- Ti6 for 10 days and 20 days and EDS results. Fig. 7 H&E staining and Giemsa staining images of the Ti6 and Ag7ZnO3HA-Ti6 implants to assess inflammatory and bacterial infection of soft tissues and bone tissues after 2 weeks and 5 weeks post-surgery.

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Fig. 8 (a) 2D and 3D micro-CT images showing new bone formation around the Ti6 and Ag7ZnO3HA-Ti6 samples implanted into the rabbit femur for 5 weeks; (b) Quantitative new bone model based on 3D micro-CT images; (c) New bone tissues evaluated by Van Gieson's picro fuchsin staining; (d) Histomorphometric measurement of new bone areas. The error bars indicate means ± standard deviations (*P < 0.05, **P < 0.01 and ***P < 0.001 (t test)).

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Table 1 Composition of the AgxZnOyHA-Ti6 composite coatings. Samples

Ag (wt%)

ZnO (wt%)

HA (wt%)

Ag0ZnO10HA-Ti6

0

10

90

Ag3ZnO7HA-Ti6

3

7

90

Ag5ZnO5HA-Ti6

5

5

90

Ag7ZnO3HA-Ti6

7

3

90

Ag10ZnO0HA-Ti6

10

0

90

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Scheme 1. Schematic illustration of fabrication process of the Ag/ZnO/HA composite coatings on Ti6Al4V as well as the bacteria killing and osteogenesis processes.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

45



Schematic illustration of fabrication process of the Ag/ZnO/HA composite coatings on Ti6Al4V as well as the bacteria killing and osteogenesis processes


Page 46 of 46

ZnO-Incorporated Hydroxyapatite Composite Coatings

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