Fabrication of Antibacterial and Antiwear Hydroxyapatite Coatings via

ACS Appl. Mater. Interfaces , 2017, 9 (5), pp 5023–5030. DOI: 10.1021/acsami.6b15979. Publication Date (Web): January 30, 2017. Copyright © 2017 Am...
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Fabrication of Antibacterial and Antiwear Hydroxyapatite Coatings via In Situ Chitosan-Mediated Pulse Electrochemical Deposition Ling Yan,†,# Yi Xiang,‡,# Jia Yu,‡ Yingbo Wang,*,† and Wenguo Cui*,‡ †

College of Chemical Engineering, Xinjiang Normal University, Urumqi, 830054, Xinjiang China Department of Orthopedics, the First Affiliated Hospital of Soochow University, Orthopedic Institute, Soochow University, 708 Renmin Road, Suzhou, Jiangsu 215006, P.R. China

‡

S Supporting Information *

ABSTRACT: Although bioinert titanium has been widely applied in orthopedics and related fields, its usage is limited by its unsatisfying osteoinductivity, anti-infection capability, and wear-resistance. Osteoinductive apatite coating can be fabricated on a titanium surface by electrochemical methods, but this causes bacterial adhesion and poor wear-resistance. On the basis of pulse electrochemical technology, a wear-resistance and antibacterial osteoinductive coating was fabricated through codeposition of hydroxyapatite (HA) and nano-Ag effectuated by the cohybridization ofchitosan (CS) with Ag+ and Ca2+. A composite coating formed with uniformly dispersed spherical nanoparticles was obtained at optimized deposition potential, Ag concentration, and apatite concentration. The nanocomposite coating shows excellent bioinductive activity; it promotes preferential growth on the (002) face, and needle-like ordered arrangement of apatite. Due to the mediation of CS hybridization, a compact structure is achieved in the HA/Ag composite coating which significantly enhances the wear-resistance of the coating and reduces the release of Ca2+ and Ag+. The antibacterial rate of the coating on Escherichia coli and Staphylococcus aureus is up to 99% according to the antibacterial test. In conclusion, a wear-resistant and long-term antibacterial bioactive nanocomposite coating is successfully fabricated on titanium surface through the strategy established in this study. KEYWORDS: hydroxyapatite, composite coating, pulse electrochemical deposition, antibacteria, frictional wear

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INTRODUCTION Medical titanium material has been widely used in plastic surgery and dental implantation over the past decade; yet metallic materials are incapable of inducing bone regeneration due to their bioinertness.1,2 One approach to improve the osteoinductive capability of the metallic implants is to fabricate apatite coatings on their surfaces. Unfortunately, while enhancing osteoinductivity, apatite coatings also promote bacterial adhesion, which aggravates the infection of the implant and therefore results in the implantation failure. Silver has good antibacterial properties that it suppresses both Grampositive and Gram-negative bacterium, and is subjected to very low resistance in bacterium.3 Hence, embedding silver nanoparticles (Ag-NPs) in the apatite coatings is an efficient approach to enhance the long-term antibacterial effect of the coatings. Common methods to embed silver in apatite coatings include plasma-spraying,4 ion-beam-assisted deposition,5 and magnetron sputtering,6 all of which are linear technologies and could not be applied on complicated-shaped medical materials. When prepared with microarc oxidation7 and sol−gel method,8 the Ag-NPs loaded in the apatite coatings tend to aggregate, leading to an increase in cytotoxicity due to exceeded local silver concentration. Therefore, uniform dispersal of Ag-NPs is the key point in the fabrication of a silver-loaded apatite coating © 2017 American Chemical Society

with long-term antibacterial capability. Furthermore, after implantation, the debris produced by wear usually causes an adverse reaction to the surface of the implant, and thus results in aseptic loosening and implant failure. For these reasons, establishing a wear-resistant as well as long-term antibacterial apatite coating becomes an urgent challenge. Studies on biomedical ceramics mainly focus on bioactive hydroxyapatite (HA) ceramics, for HA is a vital component of the human skeleton.9 Current methods to fabricate HA coatings include plasma-spraying,10 laser-clad,11 ion implantation,12 sol−gel method,13 hydrothermal deposition,14 microarc oxidation,15 electrochemical deposition,16 and so forth. Nevertheless, there are shortcomings in these methods such as pyrolysis, poor crystallinity of HA and unsatisfying binding ability with the matrix. Under mild conditions electrochemical deposition17 is a simple technique in which the loss of hydroxyl group, phase transition and brittle fracture of HA caused by thermal spraying could be effectively avoided. Coatings fabricated in this way are free from residual thermal stress, enhancing their adherence the matrix. Additionally, electronReceived: December 13, 2016 Accepted: January 17, 2017 Published: January 30, 2017 5023

DOI: 10.1021/acsami.6b15979 ACS Appl. Mater. Interfaces 2017, 9, 5023−5030

Research Article

ACS Applied Materials & Interfaces chemical deposition can be used to coat a matrix having complicated shapes and a porous surface. This is not possible for spraying methods. However, large amounts of hydrogen are by-produced upon the deoxidation of water on cathode surfaces, heavily blocking the nucleation and growth of HA, which makes the coating film loose and porous with inferior adhesive strength. Therefore, the problem remains here is how to bind HA coating and titanium surface firmly together through electrochemical deposition. Recent efforts on surface functionalization of titanium involve not only molecule immobilization on the surface but also prevention against inflammatory reaction of host tissue and bacterial infection. Enduing titanium surface with antibacterial properties is of great clinical significance. Although it is common to immobilize antibiotics on material’s surface, especially for HA surfaces, previous works found antibiotics are completely released a few days after implantation. Li et al.18 ascertained that the antibacterial effect of the titanium surface was duet to gentamicin absorbed on its electrospun coating; however, 70% of the gentamicin was released after 14 h. Vancomycin-absorbed HA surface fabricated by Tu et al.19 released over 90% of the vancomycin in 100 h. The antibacterial effect provided by sustained release of antibiotics by aid of physical encapsulation and chemical cross-linking rarely lasted for more than 5d. Besides, increasing resistance in bacteria to antibiotics is also a major concern. Therefore, a highly combinable HA coating with long-term antibacterial activity is urgently demanded. Pulse electrodeposition is the predominant approach to control the reaction rate. Previously, gelatin-loaded HA coating on titanium fabricated through pulse electrodeposition was found to be uniform in structure and well-crystallized. The intermittent reaction on the electrodes because of constantly repeated “on−off−on” pulse process in the electrolytic cell meant that when a pulse is exerted benefits the diffusion process and reduces concentration polarization; thus the deposition rate is boosted and advanced deposition effect is achieved. On this basis, we designed a pulse electrochemicaldriven Chitosan (CS)-based in situ mediation strategy to realize HA nanoparticles(HA-NPs)/Ag-NPs codeposition and bilayer regulation. The CS-Ag+ complex formed during the electrochemical driving process moves toward the negatively charged pore under the effect of an electric field, which slows the deposition of Ag+, allowing it to disperse uniformly on the negative surface. Electrostatic repulsion between CS molecules also distributes Ag-NPs in the coating by preventing their aggregation. Meanwhile, double-release is implemented by hybridization of CS and Ca2+ in HA at the microscale level. Scheme 1 is a paradigm of the mechanism of CS-mediated formation of HA-NPs and Ag-NPs. Therefore, uniformly codeposited HA-NPs and Ag-NPs fabricated by pulse electrochemical-driven CS-based in situ mediation will hopefully result in an antibacterial/antiwear dual-functional composite coating. In this study, CS, used as an Ag ion stabilizer, and pulse electrodeposition are utilized to fabricate an antiwear and longterm antibacterial HA coating, and the dual mechanism of CS’s stabilization effect on Ag-NPs and HA-NPs is revealed. The effect of deposition potential and concentration of silver and calcium on the wear resistance and antibacterial performance is systematically investigated. The bioactivity, physiological stability, and antibacterial and wear-resistant properties of the coating are also observed. Thus, a simple strategy to fabricate

Scheme 1. CS-Mediated Simultaneous Formation Mechanism of HA-NPs and Ag-NPs by Pulse Electrochemical Synthesis

an antiwear and antibacterial coating on titanium surfaces is established.

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EXPERIMENTAL SECTION

Materials. Ca(NO3)2·4H2O, (NH4)H2PO4, AgNO3, Chitosan (Medical, Sigma, Mw = 50 × 104, deacetylation degree: 91%), pure titanium (Baoji INT Medical Titanium Co., Ltd. Purity> 99.9%), other chemicals are analytical grade. Characterization Methods. X-ray powder diffractormeter (XRD, D2-PHASER, BRUKER, CuKα, 40 kV, scanning rate: 2°/min, scanning radius: 20−50°) was used for the phase composition and crystallinity of the samples. Fourier Transform Infrared Spectroscopy (FT-IR, TERSOR27, BRUKER, wavenumber region: 400−4000 cm−1) was used for the compositional changes of the samples; Scanning electron microscope (SEM, LEO-1430VP, Zeiss, Germany) was used for morphologies and sizes of the resulting products. Atomic absorption spectrometer (AAS, Z-2000, Hitachi, Japan) was used for the concentrations of each ion in the solution. Electrochemical workstation (CHI660, Shanghai Chenhua Instrument Co., Ltd.) was used to prepare samples. Ultrafunctional attrition testing machine (CETR UMT-3, Bruker, GER) was used for tribological properties of the samples. Fabrication of HA/Ag Composite Coating Mediated by CS. Pretreatment of the titanium surface: 9 × 9 × 1 mm3 medical titanium plate was selected as the matrix. The composite coating tends to come off under the impact of oxidizing the film on the titanium surface. Therefore, pretreatment of the titanium plate is necessary. The plate was deoiled by acetone, and ultrasonic cleaned for 10 min 3 times, 10 min per round. Then the plate was eroded by a chemical mixture (VH2SO4:VHCl:VH2O = 1:1:1) under 60 °C for 3−5 h, followed by heating in activating solution under 60 °C for 1 h. The plate was ultrasonic cleaned with deionized water after each step. Experimental Procedure. To prepare the electrolyte solution, Ca(NO3)2·4H2O and NH4H2PO4 (n[Ca]/[P] = 1.67) was dissolved in distilled water. A certain amount of AgNO3/CS premixture was added afterward. The pH was adjusted to 5.0 with ammonia. CS concentration was maintained at 0.33 g·L−1 throughout the process. The cathode was titanium, the anode was platinum, and the reference electrode was saturated calomel electrode. The titanium sample was placed in the three-electrode electrolytic cell for controlled-potential electrodeposition at room temperature. The conduction time was 7200 s, and the pulse width was 100 s. The sample was washed with deionized water and air-dried immediately after deposition. Bioactivity Test. The composite coating was soaked in the supersaturated calcium phosphate solution (SCPS) at 37 °C which was prepared with Tris-HCl as a buffer and regulated to pH 6.2−6.3 at room temperature; the ion concentration of which was identical with that of blood (Table 1). The SCPS was replaced every 24 h. The sample was removed from the solution 10 days after soaking, washed with deionized water, and air-dried. Friction Properties Test. Reciprocating sliding tribological tests were conducted on HA/Ag, HA/Ag/CS coatings and a pure titanium sample as control. Normal load (Fn) was 5 N and 10 N; displacement amplitude was 2 mm; f (frequency) = 0.5 Hz; t (friction test time) = 5024

DOI: 10.1021/acsami.6b15979 ACS Appl. Mater. Interfaces 2017, 9, 5023−5030

Research Article

ACS Applied Materials & Interfaces Table 1. Ion Concentrations of Supersaturated Calcium Phosphate Solution ions −1

concentrations (mmol·L )

Na+

Ca2+

Cl−

HPO42−

142.0

12.5

217.0

5.0

30 min; zirconium oxide ceramic balls (diameter = 11 mm, hardness = HV50g1800, Ra (roughness) = 0.03 μm) was utilized as friction pair. Ion Release Test. The composite coatings were soaked in 40 mL phosphate buffer (PBS, pH = 7.4) at 37 °C for 10 d to investigate the release profile of Ag+ and Ca2+. The concentration of Ag+ and Ca2+ in the lixivium was calculated according their standard curve and their absorbance was measured by AAS (Table 2). Physiological stability of the coatings was determined accordingly. Antibacterial Test. The antibacterial performance of the coatings against Gram-positive bacterium (Staphylococcus aureus) and Gramnegative bacterium (Escherichia coli) was investigated. Spread plate method and film adhering methods were applied for qualitative and quantitative analysis, respectively. The experiment was conducted on three groups: HA (as control), HA/Ag, and HA/Ag/CS, with three identical samples in each group. Qualitative Analysis. Escherichia coli and Staphylococcus aureus were separately inoculated on LB medium and activated at 37 °C for 12 h before seeding into fluid mediums and incubating for 12 h to obtain bacterium suspensions. The suspensions were further prepared into 1 × 108cell·mL−1 suspensions (10 mL) with PBS separately, which were incubated in a shaker (37 °C, 200 r·min−1) for 12 h, and then shaken well for plate spreading. The plates were cultivated in a constanttemperature incubator (37 °C) for 12 h. The growth of the bacteria was observed and photographed. Quantitative Analysis. The antibacterial ratio of the samples was tested by film adhering method. Both strains were inoculated on LB medium and cultivated (37 °C, 12 h) for three times to obtain pure colonies. Single colonies were seeded in fluid mediums and incubated in a shaker (37 °C, 200 r·min−1) for 12 h before further prepared into 3.0 × 107cell·mL−1 suspension (10 mL) with PBS, and diluted 3 times. The samples were placed on a slide in a culture dish, with sterile water on the bottom to protect the bacterium solution from evaporation. 50 μL suspension was dripped on the sample and cultivated at 37 °C for 12 h. After that, the solution was transferred into a 500 μL PBS solution and shaken well for plate smearing. The plates were cultivated for 12 h, and number of bacterial colonies were obtained. The antibacterial performance is demonstrated through the antibacterial ratio (computation method shown below). The experiment was conducted on three groups: HA (as control), HA/Ag, and HA/Ag/CS with three parallel samples in each group to obtain the average antibacterial ratio. antibacterial ratio =

Figure 1. HA/Ag/CS composite coating fabricated at different deposition potential. (a) −1.2 V of SEM; (b) −1.3 V of SEM; (c) −1.2 V and −1.3 V of XRD analysis; (d) −1.3 V of FTIR; (e) −1.3 V of TG; (f) −1.3 V distribution diagram of elements; (g) Ag element of EDX; (h) Ca element of EDX; and (i) P element of EDX analysis.

layer (Figure 1a,b) when the pulse potential is −1.2∼−1.3 V. When the potential rises to 2.0 V, the coating surface becomes bare and plenty of floccule emerges at the bottom of the beaker. This is because the reaction happens mildly when the current density, which is triggered by relatively low pulse potential, is weaker. This allows complete chelation between CS and Ag+ or Ca2+ and slows the deposition of HA-NPs and Ag-NPs. In this way, the nucleus has sufficient time for electro-crystallization, and HA-NPs and Ag-NPs have the same growth rate, resulting in the morphological transition of the particles on the surface into nanospheres, while plenty of floccules are formed due to the excessive current density on the cathode surface at high pulse potential, which makes the reaction happen too fast. Therefore, it is inadvisible to set the pulse potential excessively high. HA particle size plays a crucial role in cell adhesion and activity. Research shows that HA-NPs can increase protein adsorption and cell adhesion to improve the biological properties;20,21 thereby improving the physical and chemical properties of the coating, which results in a biomedical material with good comprehensive performance. As indicated by XRD analysis (Figure 1c), the apatite obtained when the pulse potential is −1.2 V is poorly

CCCG − CCEG × 100% CCCG

where CCCG is the colony count of the control group and CCEG is the colony count of the experimental group. Statistical Analysis. All data are presented as means ± standard deviation. Statistical analysis was carried out using One Sample t Test (assuming unequal variance). The difference between two sets of data was considered statistically significant when P < 0.05.

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RESULTS AND DISCUSSION Effects on the CS-Mediated Composite Coatings. As illustrated in Figure 1, the composite coating is formed with uniformly dispersed spherical nanoparticles covered with a CS

Table 2. Working Parameters of Atomic Absorption Spectrometer element

wavelength (nm)

bandwidth spectrum (nm)

lamp electric current (mA)

air pressure (MPa)

ethyne of flux (L·min−1)

Ca Ag

422.7 328.1

0.5 1.3

5 9

0.2 0.16

1.2 15

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DOI: 10.1021/acsami.6b15979 ACS Appl. Mater. Interfaces 2017, 9, 5023−5030

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ACS Applied Materials & Interfaces crystallized HA in the coating, while the apatite obtained at −1.3 V was well crystallized HA in the coating. Ban et al.22 suggest that the deposition of calcium and phosphorus is dependent on the OH− produced by the cathode. When the potential is relatively low, OH− is produced constantly as the electrochemical reaction takes place on the cathode, which significantly increases the supersaturation of the solution near the cathode, depositing calcium and phosphorus to form apatite. Increase in the potential makes the reaction violently with OH− produced in large quantities and diffused into regions near the cathode. As a result, calcium and phosphorus are deposited before being diffused to the cathode surface, and the deposition process turns into homogeneous nucleation in the solution instead of heterogeneous nucleation on the cathode surface, reducing the deposition rate on the coating. Hydrogen evolution on the cathode is another factor that can influence the coating deposition. A hydrolysis reaction, which produces hydrogen, occurs sharply at high potential, preventing the deposition of calcium and phosphorus, which has an impact on the morphology and crystallization of the coating. Figure 1d is the FTIR spectrum of the composite coating. The peaks of pure CS are assigned as follow: peaks for stretching vibration of CH in the aliphatic chain of CS are observed at 2932 and 2896 cm−1;23 the peak of the characteristic group, −NH2, appears at 1599 cm−1,24 while the same peak shows up at 1571 cm−1 in the HA/Ag/CS spectrum. The 607 and 560 cm−1 peak are attributed to the bending vibration of PO43−. The CS characteristic −NH2 peak in the coating spectrum is blue-shifted compared to that in pure CS, because of the hybridization of N and Ca.25 Ag-NPs tend to agglomerate because of the high deposition rate of Ag+ during electrodeposition, which causes excessive local silver content within the coating, causing slight toxicity which affects the activity of cells. Therefore, the uniform distribution of Ag-NPs is essential. As Figure 1g shows, silver is uniformly dispersed on the surface of the titanium matrix, accounting for 2.0 wt % of the coating. Calcium and phosphorus are also dispersed uniformly in the coating. Previous work indicates that CS acted as a stabilizer in the coating to ensure the uniform dispersion of Ag-NPs and HANPs. In HA/Ag/CS composite coatings, HA and Ag in the inorganic phase are stable at 600 °C,26 while the principle chains of CS begin to fracture at 210 °C and are completely decomposed at 600 °C.27 According to the thermo gravimetric analysis showed in Figure 1e, the mass of residual inorganic phase after the complete decomposition of CS was 38.6%. Therefore, the content of chitosan is 61.4%. On the basis of the analysis above, the coating fabricated at low pulse potential (−1.3 V) is well distributed and crystallized. Effects on the CS-Mediated Composite Coatings of Ag+ Concentration. Figure 2 indicates that the concentration of Ag+ has a slight impact on the morphology of the composite coating. The coating shows up as nanospheres which uniformly cover the titanium matrix in SEM photos when the Ag+ concentration is between 0.02−0.06g·L−1because CS can completely chelate with Ag+ in this range, ensuring that AgNPs are deposited on the matrix surface uniformly and ordered covered with a CS film (Figure 2a−c). Ag+ concentration has less impact on the particle size of the nanospheres (Figure 2d), but it affects their composites. According to Figure 2e, the HA diffraction peak is weak when Ag+ concentration is relatively low (0.02−0.04g·L−1); while the diffraction peak of HA appears when Ag+ concentration is higher (0.06g·L−1), with a strongest

Figure 2. HA/Ag/CS composite coating fabricated with different Ag+ concentration. (a) 0.02 g·L−1 of SEM; (b) 0.04 g·L−1 of SEM; (c) 0.06 g·L−1 of SEM; (d) particle size distribution; and (e) XRD analysis.

elemental diffraction peak of Ag. The (111) face at 38.1° is identical with the standard spectra (#04-0773). These results suggest that 0.06g·L−1 is a beneficial concentration for HA-NPs and Ag-NPs to co-deposit. Effects on the CS-Mediated Composite Coatings of Ca2+ Concentration. The concentration of Ca2+ is influential to the morphology of the composite coating. Ideal nanospheres are obtained when Ca2+ concentration is 5.0 mmol·L−1 (Figure 3a) with uniform dispersion and CS film coverage, when CS

Figure 3. HA/Ag/CS composite coating fabricated with different Ca2+ concentration. (a) 5 mmol·L−1 of SEM; (b) 16.7 mmol·L−1 of SEM; (c) particle size distribution; and (d) XRD analysis.

and Ca2+ are completely chelated to deposit HA-NPs on the titanium matrix uniformly. When Ca2+concentration increase to 16.7 mmol·L−1 (Figure 3b), the nanoparticles are still spherical and uniformly distributed, with a small amount of CS between the particles. Ca2+ concentration has a slight impact on the particle size of the spheres (Figure 3c). As the XRD analysis indicates, the HA diffraction peak of the composite coating appears strong with a dicalcium phosphate (CaHPO4·2H2O, DCPD) diffraction peak at 20.9° (PDF#09-0077) at high Ca2+ concentration (16.7 mmol/L). In the FTIR analysis (Figure S1 of the Supporting Information) of the composite coating, PO43− shows a stretching vibration peak at 1035 cm−1 and two bending vibration peaks at 603 and 567 cm−1. The characteristic peaks at 526 and 887 cm−1 identify a HPO42− group.28 CS characteristic group, −NH2 shows a weak peak at 1571 cm−1, while the peak of the same group in pure CS appears at 1599 5026

DOI: 10.1021/acsami.6b15979 ACS Appl. Mater. Interfaces 2017, 9, 5023−5030

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ACS Applied Materials & Interfaces cm−1,24 suggesting that CS characteristic −NH2 peak in the coating spectrum is blue-shifted compared to that in pure CS, because of the hybridization of N and Ca.25 On the basis of the XRD and FTIR data, it is confirmable that the inorganic phase in the composite coating is HA and DCPD. To conclude, −1.3 V pulse potential, and 0.06g·L−1 Ag+ with 5 mmol·L−1 Ca2+ is the optimal experimental condition in this study. Bioacitivty, physiological stability, antibacterial performance, and friction property analysis are conducted under this condition. Bioactivity. As shown in Figure 4, both HA/Ag composite coating and HA/Ag/CS composite coating are spherical and

coating and the HA/Ag/CS composite coating are of good bioactivity. Friction Properties. The friction coefficient curve of the coating under different normal loading is illustrated in Figure 5a,e. From the curve we can see major fluctuation takes place on the pure titanium curve in a significantly shortened period as the normal loading rises. The smooth curve in the early stage of wear is probably due to the protection of the oxide layer of the titanium surface. However, aggravation of the wear makes the wear-debris produced by the damaged oxide layer involved in the friction and wear process, leading to the obvious fluctuation of the friction coefficient curve. The friction coefficient curve of HA/Ag coating shows a substantial jump when the friction begins, followed by a sudden fall after a few minutes, and then rises with fluctuation, which is distinctly different from that of pure titanium. As for the HA/Ag/CS coating, the friction coefficient curve keeps steady at low loading. Morphological analysis is conducted on a scratch. For pure titanium, when the normal loading is 5N, abrasion furrows are obvious in the middle of the scratch, the major wear mechanism of which is abrasive. As the normal loading rises to 10N, the major mechanism becomes fatigue wear and delamination wear (Figure 5b,f). The wear mechanism of HA/ Ag composite coating is similar to that of pure titanium. Few changes take place on morphology and characteristics of the scratch, suggesting the improvement in friction properties that HA/Ag composite coating has on titanium has its limits. As for the HA/Ag/CS composite coating, its wear mechanism is predominantly adhesive wear mechanism. Morphology and characteristics of the scratch are not visibly changed with the increase of normal loading (Figure 5d,h), suggesting HA/Ag/ CS composite coating are capable of promoting the friction properties of biomedical titanium. It is reported29 that the CS/ HA coating forms a lubricating film with sufficient wearresistance on the surface under physiological conditions, as hydrated ions are formed with the phosphate anions in the coating and turn into a large hydration layer, which leads to molecular ball-bearings lubricated contact to enhance the antifriction property of the coating. Physiological Stability. As we can see from Figure 6, on the 10th day of release in PBS, the release ratio of Ca2+ of HA/ Ag was 58.6% and Ag+ 42.2%. Release of both ions shows a significant decrease with the addition of CS in the coating, which is 42.2% for Ca2+ and 13.6% for Ag+, due to the hybrid composite with Ca2+ from HA on microscale and chelation with

Figure 4. Mineralization data diagram of the composite coatings. (a) SEM photo of HA/Ag/CS composite coating; (b) SEM photo of HA/ Ag composite coating; and (c) XRD analysis.

nanosized before mineralization, and become coated by orderly ranged needle-like deposition after mineralization. The deposited HA, showing a markedly increase in the intensity and number of diffraction peak compared to that before mineralization. The mineralized coating grows preferentially on the (002) face at 25.8°, or along the z-axis. These results indicate that the bioactivity of HA/Ag composite coating is not reduced by the addition of CS. Both the HA/Ag composite

Figure 5. (a) Friction coefficient curve when normal loading is 5N; (b)Ti (Fn = 5N); (c) HA/Ag (Fn = 5N); (d) HA/Ag/CS (Fn = 5N); (e) friction coefficient curve when normal loading is 10N(f) Ti (Fn = 10N); (g) HA/Ag (Fn = 10N); and (h) HA/Ag/CS (Fn = 10N). 5027

DOI: 10.1021/acsami.6b15979 ACS Appl. Mater. Interfaces 2017, 9, 5023−5030

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ACS Applied Materials & Interfaces

enzymes. Therefore, Gram-positive bacteria (Staphylococcus aureus) on the antibacterial drug resistance stronger.31 Both qualitative and quantitative results indicate that HA/Ag/CS composite coating has excellent antibacterial performance on Escherichia coli and Staphylococcus aureus. Ag-NPs in the composite coating enhance its antibacterial performance. The major antibacterial mechanism of Ag32,33 is photocatalysis and its impact as metal ion. The high catalytic capability of Ag is determined by its chemical structure. Ag at high oxidation state has an extremely high reduction potential that makes the space around it produce atomic oxygen, which is strongly oxidizing and kills bacteria. Besides, Ag+ has a very strong attraction to the sulfhydryl group (−SH) on the protease of bacteria to deactivate it, leading to the death of the bacteria. After that, Ag+ is freed from the dead bacteria and contacts with other colonies to repeat the process above, which is the reason why Ag is persistent in antibacteria. The CS in the composite coating is also antibacterial to a certain extent. HA/ Ag/CS composite coating in the human body weak acid environment CS easy to dissolve, dissolved solution containing amino (−NH3+). It is generally assumed that the polycationic nature of chitosan, conveyed by the positively charged −NH3+ groups of glucosamine, might be a fundamental factor contributing to its interaction with negatively charged surface components of many fungi and bacteria, causing extensive cell surface alterations, leakage of intracellular substances, and ultimately resulting in impairment of vital bacterial activities.34 Additionally, CS has a strong capability to bind with metal, especially with Ag, to reduce the toxicity of Ag.35 It is reported that composite coating with 2.1 wt % of Ag shows significant inhibition zone on Escherichia coli.36 In this study, the antibacterial ratio of the composite coating containing 2.0 wt % Ag is up to 100%. Despite its excellent antibacterial performance, the HA/Ag/CS composite coating does not affect the adhesion and proliferation of cells. 2.4 wt % of Ag in composite coating is reported to promote the proliferation and differentiation of bone marrow stromal cells (BMSCs) without cytotoxicity. The Ag content in this study is 2.0 wt %, which means it is not cytotoxic.37

Figure 6. Physiological stability of the composite coating. (a) Release profile of Ag+ and (b) release profile Ca2+.

Ag+ of CS to reduce the release ratio of Ca2+ and Ag+.30 Therefore, HA/Ag/CS composite coating is physiologically stable to play the role of a long-term bacterial agent. Antibacterial Performance. Escherichia coli and Staphylococcus aureus are not only typical representative of Gramnegative and Gram-positive bacteria, but also the most common bacteria in clinical infection. It is evident from Figure 7 that

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Figure 7. Antibacterial results of the spread plate method for HA/Ag/ CS.

CONCLUSIONS Here we present a method to in situ double-mediate the codeposition of HA-NPs and Ag-NPs on titanium surface with CS, driven by pulse electrochemistry, to obtain a wear-resistant and long-term antibacterial composite coating on a titanium matrix surface. The optimized conditions of the method in this study are deposition potential −1.3 V, Ag+ concentration 0.06 g·L−1 and Ca 2+ concentration 5 mmol·L−1. Uniformly distributed spherical nanoparticles are obtained on the titanium surface. A bioactivity test shows that the composite coatings can induce the generation of apatite in fast mineralizing solution and possesses excellent bioactivity. The enhancement in the antiwear properties on the titanium matrix is proven in the friction experiment. Furthermore, the release profile demonstrates the mediation role of CS to reduce the release ratio of Ca2+ and Ag+, which increases the physiological stability of the composite coating, and the antibacterial rate of the coatings is up to 99% in the antibacterial test, indicating its good antibacterial properties. To sum up, we successfully fabricated a composite coating with CS-mediated HA-NPs and Ag-NPs that exhibited good antiwear properties and long-term antibacterial performance.

after incubation for 12 h, a lawn is formed with piled colonies on the HA coating surface in the control group, and slime can be observed on the bacteria, indicating their strong metabolism. Colony count distinctly decreases in the HA/Ag composite coating group compared to the control. No Escherichia coli and only a few colonies of Staphylococcus aureus are found on the surface of HA/Ag/CS composite coating. These results indicate that HA/Ag/CS composite coating has good antibacterial capability in bacteria solution of high concentration. Meanwhile, its antibacterial performance is quantitatively analyzed. The antibacterial rate of HA/Ag/CS coating is 100% on Escherichia coli and 99.9% on Staphylococcus aureus. The cell wall of Gram-negative bacteria (Escherichia coli) is composed of thin peptidoglycan layer (7−8 nm) with an additional outer membrane. In contrast, Gram-positive bacteria (Staphylococcus aureus) contain a thick peptidoglycan layer (20−80 nm) on the outside of the cell wall instead of outer membranes. Peptidoglycan is a mesh-like polymer consisting of sugars and amino acids. Peptidoglycan layer protects against antibacterial agents such as antibiotics, toxins, chemical, and degradative 5028

DOI: 10.1021/acsami.6b15979 ACS Appl. Mater. Interfaces 2017, 9, 5023−5030

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ACS Applied Materials & Interfaces

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b15979. FTIR spectra of the composite coatings prepared by Ca2+ concentration of 16.7 mmol·L−1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86 09914333279. E-mail: ybwang20002575@163. com (Y.W.). *Tel/Fax: +86 051267781420. E-mail: [email protected] (W.C.). ORCID

Wenguo Cui: 0000-0002-6938-9582 Author Contributions #

These authors contributed equally.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51662038 and 11572211), Science and Technology Innovation Project of Xinjiang Normal University (XSY201602005), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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REFERENCES

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