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Preparation of vancomycin-loaded alginate hydrogel coating on magnesium alloy with enhanced anticorrosion and antibacterial properties
Thin Solid Films 693 (2020) 137679
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Preparation of vancomycin-loaded alginate hydrogel coating on magnesium alloy with enhanced anticorrosion and antibacterial properties ⁎
Du Mintinga, Huang Linlina, Peng Mengkea, Hu Fenyana, Gao Qiangc, Chen Yashaoa, , Liu Pengb,
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a Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China b Key Laboratory of Biorheological Science and Technology of Ministry of Education, Chongqing Key Laboratory of Soft-Matter Material Chemistry and Function Manufacturing, College of Bioengineering, Chongqing University, Chongqing 400044, China c Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, Xi'an Jiaotong University, Xi'an 710049, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Magnesium alloy Hydrogel coating Surface modification Glycopeptide antibiotic Anticorrosion Antibacterial property Hemocompatibility
In this study, vancomycin (Van)-loaded sodium alginate (SA) hydrogel coating was fabricated on micro-arc oxidation (MAO)-treated magnesium alloy (MgA) surface via the electrostatic interaction between polyethyleneimine and SA to enhance anticorrosion, hemocompatibility, and antibacterial properties of MgA substrate. The surface morphology and chemical composition of the modified MgA substrate were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. The results showed the successful preparation of Van-loaded SA hydrogel coating on MgA/MAO substrate. Furthermore, the anticorrosion property of the coated MgA in simulated body fluids was also evaluated. The potentiodynamic polarization test indicated that corrosion resistance of MgA coated with the Van-loaded SA hydrogel was significantly improved. Biocompatibility of the modified MgA substrates was investigated by in vitro platelets adhesion assay, hemolysis ratio test, and dynamic coagulation time test. The results indicated that the modified MgA exhibited excellent antibacterial property and hemocompatibility. This study may provide an alternative pathway for surface modification of MgA implants to enhance their corrosion resistance and biocompatibility.
1. Introduction As medical and implantable metallic materials, magnesium (Mg) and its alloys have been extensively applied in orthopedic surgeries due to their excellent biodegradability, biocompatibility, and their mechanical properties similar to those of natural bone [1–3]. However, their susceptible corrosion in the physiological environment has limited the clinical application of Mg alloy (MgA) [4–8]. Furthermore, MgA faces implant-related infection, which leads to implant failure, complications, morbidity, and mortality in the human bodies [9–12]. Currently, surface modification has been an effective strategy to enhance anticorrosion and antibacterial properties of Mg and its alloys without affecting their intrinsic properties. To protect the Mg-based implant materials from rapid corrosion and improve their biocompatibility in vivo, natural polymer-based coatings have attracted extensive attention [13–15]. Sodium alginate (SA) is a type of natural polymers, which has been widely applied in tissue engineering as a controlled release vehicle for drug delivery [16–18], due to its potential advantages such as excellent biocompatibility and
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biodegradability. The application of SA hydrogel as coatings on MgA surface was reported by Kumta et al. [19], wherein hydroxyapatite loaded SA hydrogel-coating effectively enhanced the bioactivity of MgA substrate. However, the preparation of drug-loaded hydrogel coating on the MgA surface has rarely been reported. In our previous studies, chitosan and poly(styrene sulfonate) polyelectrolyte multilayers, polymerized 2-methacryloyloxyethyl phosphorylcholine, and poly(ethylene glycol) methacrylate coatings were fabricated on micro-arc oxidation (MAO)-treated MgA surface for enhancing anticorrosion performance and hemocompatibility [20–22]. However, apart from anticorrosion and hemocompatibility, antibacterial properties of MgA should also be paid attention to and taken into account. Vancomycin (Van), a water-soluble and little toxic glycopeptide antibiotic, is usually used as the “drug of last resort” for the treatment of lethal infections caused by methicillin-resistant staphylococcus aureus (MRSA) [23]. It has been reported that a Van-loaded dual-function injectable hydrogel can kill most of MRSA (>99.999%) when the bacteria come in direct contact with the gel [24]. Moreover, Van-coated titanium alloy prepared by Zhang et al. showed excellent
Corresponding authors. E-mail addresses: [email protected] (Y. Chen), [email protected] (P. Liu).
https://doi.org/10.1016/j.tsf.2019.137679 Received 18 September 2018; Received in revised form 19 September 2019; Accepted 29 October 2019 Available online 31 October 2019 0040-6090/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic diagram of the preparation process of MgA/MAO/cross-linked SA-Van.
The static WCA was averaged at least five points of measurements and calculate the average value.
antibacterial property against MRSA and good cytocompatibility to human osteoblast cell line MG-63 [25]. Herein, a Van-loaded SA hydrogel coating was fabricated on the surface of MAO-treated MgA substrate to improve its anticorrosion, hemocompatibility, and antibacterial properties. The overall preparation process of coating is illustrated in Fig. 1. The influence of such surface modification on corrosion, hemocompatibility, and antibacterial properties was investigated in vitro as well.
2.4. Electrochemical corrosion test The electrochemical corrosion was performed to investigate the corrosion behavior of the samples using an electrochemical workstation (CS350, Wuhan Corrosion Test Instrument Co. Ltd.). The electrochemical corrosion test was performed in a standard simulated body fluid (SBF) at 36 ± 0.5 °C [27]. A three-electrode system (MgA (working), platinum plate (counter) and saturated calomel electrode (reference)) was used to carry out the measurements. Prior to the potentiodynamic polarization test, the samples were stabilized in SBF for 5 min. The recording range was from −3 to 1 V of the open-circuit potential and the scanning rate was 0.05 V/s. With Tafel extrapolation methods, the corrosion potentials Ecorr and corrosion current density icorr were obtained from the potentiodynamic polarization curves.
2. Experimental methods 2.1. Preparation of MAO coating on MgA surface The MAO coating was prepared according to our previously reported procedures [26]. Briefly, the polished MgA (AZ31D) and graphite rod acted as anode and cathode, respectively. For MAO process, MgA substrate was directly treated by a pulse power source in an aqueous solution containing 5.684 g/L Na2SiO3•9H2O and 0.800 g/L NaOH for 5 min at 5 °C. The duty cycle was 30%. Pulse frequency and voltage were controlled at 100 Hz and 400 V, respectively. Then, the treated MgA samples were rinsed with distilled water. Finally, the samples dried at 60 °C and labeled as MgA/MAO.
2.5. The Van release test Briefly, MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van were immersed in 5 mL phosphatic buffer solution (PBS) at 37 °C for different time intervals. The immersion solution of the sample was taken out at the desired time. The absorbance was then measured at a wavelength of 281 nm with an ultraviolet-visible spectrophotometer. The solution was recovered, soaked until the next measurement time, and the absorbance was measured until the end of release. Substitute the measured absorbance value into the standard curve to obtain Van concentration, and finally plot the concentration-time curve.
2.2. Preparation of Van-Loaded SA hydrogel coating on MgA/MAO surface Taking advantage of the electrostatic interaction between polyethyleneimine (PEI) and SA, an ionically cross-linked Van-loaded SA hydrogel coating was prepared on MgA/MAO surface. Firstly, the MgA/ MAO substrate was dipped in a 25 mg/mL PEI solution for 30 min and rinsed with deionized water. Subsequently, the treated MgA/MAO substrate was immersed into 2 wt% SA/Van solution (the concentration of Van is 0.5 wt%) for 1 h, and then rinsed with deionized water. The sample was dried under vacuum for 48 h and labeled as MgA/MAO/SAVan. For the preparation of ionically cross-linked hydrogel coating, the MgA/MAO/SA-Van substrate was dipped in a CaCl2 solution for 1 h to allow gelation of the SA solution and then dried at room temperature (labeled as MgA/MAO/cross-linked SA-Van).
2.6. Platelets adhesion assay Platelets adhesion assay was used to assess the hemocompatibility of different MgA samples. The procedure for platelets adhesion assay was similar to previous work [28-30]. 50 μL of fresh platelet-rich plasma was added onto the surface of different MgA samples. After incubation for 2 h at 37 °C, the samples were washed with PBS (pH 7.2). The adhered platelets on the surface of MgA sample were immobilized with 2.5 wt% glutaraldehyde at room temperature for 30 min. Then, the samples were dehydrated and dealcoholized using a series of ethanol/ water mixtures. The resulting samples dried at 37 °C and characterized by SEM.
2.3. Surface characterization The surface morphologies of the original MgA and modified MgA were characterized by scanning electron microscopy (SEM) (Quanta 200, FEI). The acceleration voltages and working distances for each image were 20.0 kV and 9.5–11.5 mm, respectively. The surface chemical composition of the original MgA and modified MgA were confirmed using X-ray photoelectron spectroscopy (XPS) on a ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV) (Thermo Electron Co.). Each spectrum was recorded at 20 eV pass energy with a 45° take-off angle. Static water contact angle (WCA) measurements of the samples were performed with a videobased optical system (OCA20, Dataphysisc Co.). In detail, 2 μL deionized water was added to the sample surface and WCA was measured.
2.7. Hemolysis ratio test Take 10 mL of fresh human blood and dilute with 0.9% NaCl in a volume ratio of 4:5. Place different MgA samples in 10 mL 0.9% NaCl, and keep it in a water bath at 37 °C for 30 min, then add 0.2 mL the diluted blood, gently shake it, keep it in a water bath for 60 min, then centrifuge the liquid for 10 min at 1000 r/min, take 0.2 mL of the supernatant to measure the absorbance at 545 nm with a fully automatic microplate reader. The 10 mL 0.9% NaCl + 0.2 mL the diluted blood as 2
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negative control group, and the 10 mL deionized water + 0.2 mL the diluted blood as positive control group, and three parallel samples were set for each group. 2.8. Dynamic coagulation time test 0.2 mL fresh human blood and 20 μL 0.2 mol/L CaCl2 solution were added to the surface of different MgA samples. At 5, 10, 20, 40, 60 min, 100 mL deionized water was used to flow slowly through the surface of the samples, then the solution was collected in the beaker, and the absorbance-time curve was obtained by measuring the absorbance of the solution at the 545 nm. Three parallel samples were set for each group. 2.9. Antibacterial test Kirby-Bauer disc diffusion test was used to assess the antibacterial activity of the different MgA samples against E. coli and S. aureus. Briefly, E. coli and S. aureus were cultivated in a Mueller Hinton Broth (MHB) medium and then incubated overnight at 37 °C by a shaking incubator. The overnight bacterial suspensions were then re-cultured in a fresh MHB and grown to OD600 = 0.5. The test inoculum was diluted to 1.0 × 106 CFU/mL. Then, 100 μL of suspension solution was spread on MH-Agar plate, and subsequently different MgA samples were placed on the MH-Agar plate surface. After incubation for 24 h at 37 °C, the size of the inhibition zone of different samples were measured using the optical microscopy.
Fig. 3. SEM images and EDS spectra of cross-section of (a) MgA/MAO and (b) MgA/MAO/cross-linked SA-Van.
became smooth (Fig. 2c). This change of the surface morphology can be attributed to the viscosity of SA solution. After cross-linking with Ca ions, the micropores appeared again (Fig. 2d). In the presence of divalent Ca ions, SA can be cross-linked ionically between chains to form hydrogel. The Ca ions get exchanged with Na ions, which results in binding together of adjacent chains and causing the gelation of alginate. After the crosslinking with Ca ions, the fluidity of hydrogel degrades. Therefore, it is impossible to cover the entire surface completely, and the porous structure of MAO is exposed in some area. Fig. 3 shows the thickness of MAO coating and the Van-loaded SA hydrogel composite coating. The result indicated that the thickness of coating increased when the Van-loaded SA hydrogel coating was prepared on the MgA/ MAO surface. After MAO modification, the thickness of the MAO coating was approximately 15 μm (Fig. 3a). Moreover, when the Vanloaded SA hydrogel was deposited, the thickness of the coating was further increased to 25 μm (Fig. 3b). The chemical composition of the modified MgA substrate surfaces was characterized by XPS. The wide XPS spectra of different MgA samples are shown in Fig. 4 and the chemical element composition of different MgA sample surfaces are listed in Table 1. Fig. 4a exhibits the existence of Mg1s, O1s, and C1s peaks for the original MgA sample, where O1s and C1s are attributed to unavoidable ambient air adsorption and chemical reagents, respectively. The XPS spectrum of MgA/ MAO sample is shown in Fig. 4b, exhibiting that the amount of C and Mg decreases and the amount of O increases. Furthermore, the Si2p and Si2s peaks are also observed in Fig. 4b. These results indicated that the MAO coatings were mainly composed of MgO and accampanied by MgSiO3 [26]. Fig. 4c demonstrates the appearance of N1s peak and Cl2p characteristic peak for the sample coated with SA hydrogel containing Van, while the intensity of Mg1s peaks decreases sharply. These results suggested that the Van-loaded SA hydrogel coating was deposited on the surface of MgA/MAO via the electrostatic interaction between polyethyleneimine (PEI) and SA. When the cross-linked Vanloaded SA hydrogel coating was fabricated on MgA/MAO surface, the chemical composition of the MgA surface further changed significantly. The appearance of Ca2p peak indicated the occurrence of exchange of Na+ to Ca2+ on the MgA/MAO surface and the formation of crosslinked Van-loaded SA hydrogel coating. The results of XPS analysis demonstrated the successful preparation of cross-linked Van-loaded SA hydrogel coating on the MgA/MAO surface.
3. Results and discussion 3.1. Preparation and surface characterization of the modified MgA substrates SEM was utilized to characterize the morphology of different MgA samples. Fig. 2a exhibits that the surface of original MgA is smooth and has orderly abrasive cracks, which were caused by polishing. MAO treatment led to the generation of vesicular structures with size of 2–5 μm, which were uniformly distributed on the MgA/MAO substrate (Fig. 2b). These irregularly distributed pores were formed due to spark discharge and the trapping of gas bubbles in the process of growing oxide layer [31]. Furthermore, after Van-loaded SA hydrogel was coated on the MgA/MAO surface, the resulting substrate surface
Fig. 2. SEM images of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van. 3
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Fig. 5. Water contact angles of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
Fig. 4. XPS spectra of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
The hydrophilicity of the different MgA samples was characterized by static contact angles. Compared to the original MgA substrate (Fig. 5a), the water contact angle of MgA/MAO substrate decreased from 49.0° to 10.9°. This result might be attributed to the irregular vesicular structure of MgA/MAO surface and some hydrophilic oxides such as MgO (Fig. 5b). Compared to the surface of MgA/MAO, the surface of MgA/MAO/SA-Van was more hydrophobic due to the presence of SA and Van (Fig. 5b vs Fig. 5c). After MgA/MAO/SA-Van was cross-linked by CaCl2, the water contact angle changed from 48.3° to 22.4° (Fig. 5c and d). The above mentioned results indicated that the hydrophilicity/hydrophobicity properties of the modified MgA could be tuned by different coatings.
3.2. Evaluation of anticorrosion property of the modified MgA substrate The rapid corrosion of MgA in physiological environment hampers its clinical application. Therefore, the anticorrosion effect of the coating on MgA implant materials surface is of utmost importance to their practical application [32]. Corrosion behavior of different MgA samples was studied by electrochemical corrosion tests. The potentiodynamic polarization curves are shown in Fig. 6. The relevant parameters including the icorr and Ecorr are listed in Table 2. Compared to original MgA (Ecorr = −1.595 V), Ecorr value of the MgA/MAO, MgA/MAO/SAVan and MgA/MAO/cross-linked SA-Van increased by 11, 1209 and 1281 mV, respectively. Compared to original MgA, the corresponding icorr values of MgA/MAO, MgA/MAO/SA-Van and MgA/MAO/crosslinked SA-Van samples decreased by about 2–4 orders of magnitude (Table 2). An excellent anticorrosion performance is largely dependent on a higher positive corrosion potential or a lower polarization current [33,34]. The above mentioned results indicated that all the modified MgA exhibited excellent anticorrosion performance.
Fig. 6. Potentiodynamic polarization curves of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van. Table 2 Ecorr and icorr of different MgA samples by potentiodynamic polarization test. Curves
Ecorr (V)
icorr (A/cm2)
MgA MgA/MAO MgA/MAO/SA-Van MgA /MAO/ cross-linked SA-Van
−1.595 −1.544 −0.386
6.25 × 10−4 2.27 × 10−6 1.68 × 10−8
−0.314
3.34 × 10−7
Table 1 . Chemical element compositions of different MgA samples obtained from XPS. Samples
Mg (at%)
C (at%)
O (at%)
Si (at%)
N (at%)
Na (at%)
Cl (at%)
Ca (at%)
MgA MgA/MAO MgA/MAO/SA-Van MgA /MAO/ cross-linked SA-Van
46.2 40.3 11.0 5.2
23.0 19.8 58.3 59.0
30.8 34.0 23.0 30.0
/ 5.9 1.8 0.3
/ / 4.0 2.3
/ / 1.1 0.2
/ / 0.8 0.6
/ / / 2.4
4
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Fig. 7. Van release behavior of (a) MgA/MAO/SA-Van and (b) MgA/MAO/ cross-linked SA-Van substrates in PBS buffer.
Fig. 8. SEM images of adhered platelets on (a) MgA, (b) MgA/MAO, (c) MgA/ MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
3.3. Evaluation of the Van release behavior for MgA/MAO/SA-Van and MgA/MAO/Cross-linked SA-Van substrates
significantly inhibit platelet adhesion and improve hemocompatibility. Fig. 9A shows that the hemolysis ratios of MgA, MgA/MAO, MgA/ MAO/SA-Van and MgA/MAO/cross-linked SA-Van are 1.30, 0.93, 0.96 and 0.60%, respectively. The hemolysis ratio of MgA/MAO/crosslinked SA-Van is superior to that of other samples. This indicates that the hemolysis ratio of the modified MgA and the original MgA specifies the requirement of the medical standard. When the hemolysis ratio of the samples is less than 5%, the damage to the red blood cells is very light, and hemolysis reaction does not occur. Fig. 9B shows the dynamic coagulation time of different MgA samples. Clearly, the absorbance decreases gradually with time. When the contact time with blood is the same, the absorbance value of MgA/ MAO/cross-linked SA-Van is higher than that of other samples and the curve of MgA/MAO/cross-linked SA-Van shows the slowest decline trend, indicating that the coagulation process of MgA/MAO/crosslinked SA-Van is the slowest.
Furthermore, the Van release behavior of MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van substrates was investigated. Fig. 7 shows that MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van substrates indeed exhibit similar controlled release trend. For the first 25 min, the Van gets released very quickly from two substrates. After 50 min, the release of Van from two substrates enters a plateau phase. Drug loading amounts of MgA/MAO/SA-Van and MgA/MAO/crosslinked SA-Van substrates are 0.68 and 0.36 mg per square centimeter sample, respectively. During the cross-linking process, the loaded Van released into the solution (about 0.32 mg per square centimeter sample), which can be calculated from data presented in Fig. 7.
3.4. Evaluation of the hemocompatibility of the modified MgA substrates 3.5. Antibacterial properties of the modified MgA substrates
Platelet adhesion onto the surfaces of biomaterials triggers the coagulation of blood and thrombus formation [35]. Thus, the property of anti-platelets adhesion is usually used to evaluate the hemocompatibility of biomaterials [36]. In the current study, the morphologies and numbers of the adhered platelets on surfaces of original MgA, MgA/MAO, MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van were measured by using SEM images. Fig. 8a exhibits the presence of numerous platelets adhered dendritically on the surface of original MgA. The amounts of platelets adhered on the surface of MgA/MAO (Fig. 8b) decrease obviously compared to that of original MgA. A small amount of platelets adhered on the surface of MgA/MAO/SA-Van, exhibiting good anti-platelet adhesion properties (Fig. 8c). The MgA/ MAO/cross-linked SA-Van showed the best anti-platelets adhesion property, because the adhered platelets could hardly be observed (Fig. 8d). Furthermore, it has been reported that hydrogel coating on substrate surface can enhance anti-platelets adhesion property, and the anti-platelets adhesion ability of hydrogel is affected by the zeta potential, serum proteins adsorption, chemical component and hydrophilicity of the hydrogel [37]. However, success in these fields still remains rare. Chung et al. [38] reported that the synthesized SA-hyaluronic acid hydrogels showed similar hemocompatibility with that of medical-grade polyurethane. Joseph et al. [39] reported that the unmodified polyethylene terephthalate presented thrombogenic behavior, and improved anti-platelets adhesion property after being coated with alginate dialdehyde cross-linked gelatin hydrogel. The Van-loaded SA hydrogel coating reported herein provides an example that can
Apart from the anticorrosion performance and hemocompatibility, implant-related infection is also the major reason for the failure of implant in clinical applications, accounting for high medical cost [10–12]. In this study, the inhibition zone method was selected to estimate the antibacterial activity of the modified MgA substrates against E. coli and S. aureus. The photographs of inhibition zone of original MgA and modified MgA samples are shown in Fig. 10. The original MgA (Fig. 10a) and MgA/MAO substrates (Fig. 10b) have almost no inhibition zone area to either E. coli or S. aureus, which indicates that the original MgA and MgA/MAO samples show little antibacterial activity toward either E. coli or S. aureus. In contrast, MgA/MAO/SA-Van (Fig. 10c) and MgA/MAO/cross-linked SA-Van (Fig. 10d) substrates exhibited clear inhibition zone against S. aureus. The results indicated that the Van released from the SA hydrogel coatings performed better bactericidal effect against S. aureus than that against E. coli. Haldar et al. reported that Van and its derivatives are known to be highly active against various gram-positive bacteria [40]. Actually, in the clinical application, most of the implant-related infections are caused by S. aureus [41]. Therefore, we believe that the approach presented herein is helpful to solve implant-related infections. 4. Conclusions In this study, a Van-loaded SA hydrogel coating was developed on MAO-treated MgA surfaces. The SEM and XPS results confirmed that 5
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Fig. 9. (A) Hemolysis ratio and (B) dynamic coagulation time of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
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Fig. 10. Inhibition zone photos of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SAVan, (d) MgA/MAO/cross-linked SA-Van for E. coli and S. aureus, respectively.
Van-loaded SA hydrogel coatings were prepared on the MgA/MAO surface. Furthermore, SEM measurement illustrated that the thickness of the composite coating increased to 25 μm after the hydrogel deposition. The icorr of the MgA/MAO/cross-linked SA-Van significantly decreased approximately by three orders of magnitude compared to that of original MgA sample, exhibiting higher potential for improving anticorrosive property of MgA. More importantly, the MgA/MAO/ cross-linked SA-Van were capable of improving antibacterial properties due to the introduction of Van antibacterial agent in vitro, especially for S. aureus. Moreover, the fabricated Van-loaded SA hydrogel coating showed anti-platelets adhesion, superior hemolysis ratio, and slower coagulation process, indicating excellent hemocompatibility. Overall, the developed platform herein may pave a way toward a versatile, promising strategy for improving the anticorrosive and biological performance of biodegradable MgA in biomedical field.
Acknowledgments This work is supported by the National Natural Science Foundation of China (Nos. 21773149, 51303218), State Key Project of Research and Development (No. 2016YFC1100300), the Key Research and Development Project of Shaanxi Province of China (No. 2018GY-117), Fundamental Research Funds for the Central Universities (2018CDXYSW0023), Chongqing Research Program of Technological Innovation and Application Demonstration (cstc2018jscx-msybX0299) and Innovation Team in University of Chongqing Municipal Government (CXTDX201601002) .
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Preparation of vancomycin-loaded alginate hydrogel coating on magnesium alloy with enhanced anticorrosion and antibacterial properties ⁎
Du Mintinga, Huang Linlina, Peng Mengkea, Hu Fenyana, Gao Qiangc, Chen Yashaoa, , Liu Pengb,
T
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a Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China b Key Laboratory of Biorheological Science and Technology of Ministry of Education, Chongqing Key Laboratory of Soft-Matter Material Chemistry and Function Manufacturing, College of Bioengineering, Chongqing University, Chongqing 400044, China c Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, Xi'an Jiaotong University, Xi'an 710049, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Magnesium alloy Hydrogel coating Surface modification Glycopeptide antibiotic Anticorrosion Antibacterial property Hemocompatibility
In this study, vancomycin (Van)-loaded sodium alginate (SA) hydrogel coating was fabricated on micro-arc oxidation (MAO)-treated magnesium alloy (MgA) surface via the electrostatic interaction between polyethyleneimine and SA to enhance anticorrosion, hemocompatibility, and antibacterial properties of MgA substrate. The surface morphology and chemical composition of the modified MgA substrate were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. The results showed the successful preparation of Van-loaded SA hydrogel coating on MgA/MAO substrate. Furthermore, the anticorrosion property of the coated MgA in simulated body fluids was also evaluated. The potentiodynamic polarization test indicated that corrosion resistance of MgA coated with the Van-loaded SA hydrogel was significantly improved. Biocompatibility of the modified MgA substrates was investigated by in vitro platelets adhesion assay, hemolysis ratio test, and dynamic coagulation time test. The results indicated that the modified MgA exhibited excellent antibacterial property and hemocompatibility. This study may provide an alternative pathway for surface modification of MgA implants to enhance their corrosion resistance and biocompatibility.
1. Introduction As medical and implantable metallic materials, magnesium (Mg) and its alloys have been extensively applied in orthopedic surgeries due to their excellent biodegradability, biocompatibility, and their mechanical properties similar to those of natural bone [1–3]. However, their susceptible corrosion in the physiological environment has limited the clinical application of Mg alloy (MgA) [4–8]. Furthermore, MgA faces implant-related infection, which leads to implant failure, complications, morbidity, and mortality in the human bodies [9–12]. Currently, surface modification has been an effective strategy to enhance anticorrosion and antibacterial properties of Mg and its alloys without affecting their intrinsic properties. To protect the Mg-based implant materials from rapid corrosion and improve their biocompatibility in vivo, natural polymer-based coatings have attracted extensive attention [13–15]. Sodium alginate (SA) is a type of natural polymers, which has been widely applied in tissue engineering as a controlled release vehicle for drug delivery [16–18], due to its potential advantages such as excellent biocompatibility and
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biodegradability. The application of SA hydrogel as coatings on MgA surface was reported by Kumta et al. [19], wherein hydroxyapatite loaded SA hydrogel-coating effectively enhanced the bioactivity of MgA substrate. However, the preparation of drug-loaded hydrogel coating on the MgA surface has rarely been reported. In our previous studies, chitosan and poly(styrene sulfonate) polyelectrolyte multilayers, polymerized 2-methacryloyloxyethyl phosphorylcholine, and poly(ethylene glycol) methacrylate coatings were fabricated on micro-arc oxidation (MAO)-treated MgA surface for enhancing anticorrosion performance and hemocompatibility [20–22]. However, apart from anticorrosion and hemocompatibility, antibacterial properties of MgA should also be paid attention to and taken into account. Vancomycin (Van), a water-soluble and little toxic glycopeptide antibiotic, is usually used as the “drug of last resort” for the treatment of lethal infections caused by methicillin-resistant staphylococcus aureus (MRSA) [23]. It has been reported that a Van-loaded dual-function injectable hydrogel can kill most of MRSA (>99.999%) when the bacteria come in direct contact with the gel [24]. Moreover, Van-coated titanium alloy prepared by Zhang et al. showed excellent
Corresponding authors. E-mail addresses: [email protected] (Y. Chen), [email protected] (P. Liu).
https://doi.org/10.1016/j.tsf.2019.137679 Received 18 September 2018; Received in revised form 19 September 2019; Accepted 29 October 2019 Available online 31 October 2019 0040-6090/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic diagram of the preparation process of MgA/MAO/cross-linked SA-Van.
The static WCA was averaged at least five points of measurements and calculate the average value.
antibacterial property against MRSA and good cytocompatibility to human osteoblast cell line MG-63 [25]. Herein, a Van-loaded SA hydrogel coating was fabricated on the surface of MAO-treated MgA substrate to improve its anticorrosion, hemocompatibility, and antibacterial properties. The overall preparation process of coating is illustrated in Fig. 1. The influence of such surface modification on corrosion, hemocompatibility, and antibacterial properties was investigated in vitro as well.
2.4. Electrochemical corrosion test The electrochemical corrosion was performed to investigate the corrosion behavior of the samples using an electrochemical workstation (CS350, Wuhan Corrosion Test Instrument Co. Ltd.). The electrochemical corrosion test was performed in a standard simulated body fluid (SBF) at 36 ± 0.5 °C [27]. A three-electrode system (MgA (working), platinum plate (counter) and saturated calomel electrode (reference)) was used to carry out the measurements. Prior to the potentiodynamic polarization test, the samples were stabilized in SBF for 5 min. The recording range was from −3 to 1 V of the open-circuit potential and the scanning rate was 0.05 V/s. With Tafel extrapolation methods, the corrosion potentials Ecorr and corrosion current density icorr were obtained from the potentiodynamic polarization curves.
2. Experimental methods 2.1. Preparation of MAO coating on MgA surface The MAO coating was prepared according to our previously reported procedures [26]. Briefly, the polished MgA (AZ31D) and graphite rod acted as anode and cathode, respectively. For MAO process, MgA substrate was directly treated by a pulse power source in an aqueous solution containing 5.684 g/L Na2SiO3•9H2O and 0.800 g/L NaOH for 5 min at 5 °C. The duty cycle was 30%. Pulse frequency and voltage were controlled at 100 Hz and 400 V, respectively. Then, the treated MgA samples were rinsed with distilled water. Finally, the samples dried at 60 °C and labeled as MgA/MAO.
2.5. The Van release test Briefly, MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van were immersed in 5 mL phosphatic buffer solution (PBS) at 37 °C for different time intervals. The immersion solution of the sample was taken out at the desired time. The absorbance was then measured at a wavelength of 281 nm with an ultraviolet-visible spectrophotometer. The solution was recovered, soaked until the next measurement time, and the absorbance was measured until the end of release. Substitute the measured absorbance value into the standard curve to obtain Van concentration, and finally plot the concentration-time curve.
2.2. Preparation of Van-Loaded SA hydrogel coating on MgA/MAO surface Taking advantage of the electrostatic interaction between polyethyleneimine (PEI) and SA, an ionically cross-linked Van-loaded SA hydrogel coating was prepared on MgA/MAO surface. Firstly, the MgA/ MAO substrate was dipped in a 25 mg/mL PEI solution for 30 min and rinsed with deionized water. Subsequently, the treated MgA/MAO substrate was immersed into 2 wt% SA/Van solution (the concentration of Van is 0.5 wt%) for 1 h, and then rinsed with deionized water. The sample was dried under vacuum for 48 h and labeled as MgA/MAO/SAVan. For the preparation of ionically cross-linked hydrogel coating, the MgA/MAO/SA-Van substrate was dipped in a CaCl2 solution for 1 h to allow gelation of the SA solution and then dried at room temperature (labeled as MgA/MAO/cross-linked SA-Van).
2.6. Platelets adhesion assay Platelets adhesion assay was used to assess the hemocompatibility of different MgA samples. The procedure for platelets adhesion assay was similar to previous work [28-30]. 50 μL of fresh platelet-rich plasma was added onto the surface of different MgA samples. After incubation for 2 h at 37 °C, the samples were washed with PBS (pH 7.2). The adhered platelets on the surface of MgA sample were immobilized with 2.5 wt% glutaraldehyde at room temperature for 30 min. Then, the samples were dehydrated and dealcoholized using a series of ethanol/ water mixtures. The resulting samples dried at 37 °C and characterized by SEM.
2.3. Surface characterization The surface morphologies of the original MgA and modified MgA were characterized by scanning electron microscopy (SEM) (Quanta 200, FEI). The acceleration voltages and working distances for each image were 20.0 kV and 9.5–11.5 mm, respectively. The surface chemical composition of the original MgA and modified MgA were confirmed using X-ray photoelectron spectroscopy (XPS) on a ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV) (Thermo Electron Co.). Each spectrum was recorded at 20 eV pass energy with a 45° take-off angle. Static water contact angle (WCA) measurements of the samples were performed with a videobased optical system (OCA20, Dataphysisc Co.). In detail, 2 μL deionized water was added to the sample surface and WCA was measured.
2.7. Hemolysis ratio test Take 10 mL of fresh human blood and dilute with 0.9% NaCl in a volume ratio of 4:5. Place different MgA samples in 10 mL 0.9% NaCl, and keep it in a water bath at 37 °C for 30 min, then add 0.2 mL the diluted blood, gently shake it, keep it in a water bath for 60 min, then centrifuge the liquid for 10 min at 1000 r/min, take 0.2 mL of the supernatant to measure the absorbance at 545 nm with a fully automatic microplate reader. The 10 mL 0.9% NaCl + 0.2 mL the diluted blood as 2
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negative control group, and the 10 mL deionized water + 0.2 mL the diluted blood as positive control group, and three parallel samples were set for each group. 2.8. Dynamic coagulation time test 0.2 mL fresh human blood and 20 μL 0.2 mol/L CaCl2 solution were added to the surface of different MgA samples. At 5, 10, 20, 40, 60 min, 100 mL deionized water was used to flow slowly through the surface of the samples, then the solution was collected in the beaker, and the absorbance-time curve was obtained by measuring the absorbance of the solution at the 545 nm. Three parallel samples were set for each group. 2.9. Antibacterial test Kirby-Bauer disc diffusion test was used to assess the antibacterial activity of the different MgA samples against E. coli and S. aureus. Briefly, E. coli and S. aureus were cultivated in a Mueller Hinton Broth (MHB) medium and then incubated overnight at 37 °C by a shaking incubator. The overnight bacterial suspensions were then re-cultured in a fresh MHB and grown to OD600 = 0.5. The test inoculum was diluted to 1.0 × 106 CFU/mL. Then, 100 μL of suspension solution was spread on MH-Agar plate, and subsequently different MgA samples were placed on the MH-Agar plate surface. After incubation for 24 h at 37 °C, the size of the inhibition zone of different samples were measured using the optical microscopy.
Fig. 3. SEM images and EDS spectra of cross-section of (a) MgA/MAO and (b) MgA/MAO/cross-linked SA-Van.
became smooth (Fig. 2c). This change of the surface morphology can be attributed to the viscosity of SA solution. After cross-linking with Ca ions, the micropores appeared again (Fig. 2d). In the presence of divalent Ca ions, SA can be cross-linked ionically between chains to form hydrogel. The Ca ions get exchanged with Na ions, which results in binding together of adjacent chains and causing the gelation of alginate. After the crosslinking with Ca ions, the fluidity of hydrogel degrades. Therefore, it is impossible to cover the entire surface completely, and the porous structure of MAO is exposed in some area. Fig. 3 shows the thickness of MAO coating and the Van-loaded SA hydrogel composite coating. The result indicated that the thickness of coating increased when the Van-loaded SA hydrogel coating was prepared on the MgA/ MAO surface. After MAO modification, the thickness of the MAO coating was approximately 15 μm (Fig. 3a). Moreover, when the Vanloaded SA hydrogel was deposited, the thickness of the coating was further increased to 25 μm (Fig. 3b). The chemical composition of the modified MgA substrate surfaces was characterized by XPS. The wide XPS spectra of different MgA samples are shown in Fig. 4 and the chemical element composition of different MgA sample surfaces are listed in Table 1. Fig. 4a exhibits the existence of Mg1s, O1s, and C1s peaks for the original MgA sample, where O1s and C1s are attributed to unavoidable ambient air adsorption and chemical reagents, respectively. The XPS spectrum of MgA/ MAO sample is shown in Fig. 4b, exhibiting that the amount of C and Mg decreases and the amount of O increases. Furthermore, the Si2p and Si2s peaks are also observed in Fig. 4b. These results indicated that the MAO coatings were mainly composed of MgO and accampanied by MgSiO3 [26]. Fig. 4c demonstrates the appearance of N1s peak and Cl2p characteristic peak for the sample coated with SA hydrogel containing Van, while the intensity of Mg1s peaks decreases sharply. These results suggested that the Van-loaded SA hydrogel coating was deposited on the surface of MgA/MAO via the electrostatic interaction between polyethyleneimine (PEI) and SA. When the cross-linked Vanloaded SA hydrogel coating was fabricated on MgA/MAO surface, the chemical composition of the MgA surface further changed significantly. The appearance of Ca2p peak indicated the occurrence of exchange of Na+ to Ca2+ on the MgA/MAO surface and the formation of crosslinked Van-loaded SA hydrogel coating. The results of XPS analysis demonstrated the successful preparation of cross-linked Van-loaded SA hydrogel coating on the MgA/MAO surface.
3. Results and discussion 3.1. Preparation and surface characterization of the modified MgA substrates SEM was utilized to characterize the morphology of different MgA samples. Fig. 2a exhibits that the surface of original MgA is smooth and has orderly abrasive cracks, which were caused by polishing. MAO treatment led to the generation of vesicular structures with size of 2–5 μm, which were uniformly distributed on the MgA/MAO substrate (Fig. 2b). These irregularly distributed pores were formed due to spark discharge and the trapping of gas bubbles in the process of growing oxide layer [31]. Furthermore, after Van-loaded SA hydrogel was coated on the MgA/MAO surface, the resulting substrate surface
Fig. 2. SEM images of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van. 3
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Fig. 5. Water contact angles of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
Fig. 4. XPS spectra of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
The hydrophilicity of the different MgA samples was characterized by static contact angles. Compared to the original MgA substrate (Fig. 5a), the water contact angle of MgA/MAO substrate decreased from 49.0° to 10.9°. This result might be attributed to the irregular vesicular structure of MgA/MAO surface and some hydrophilic oxides such as MgO (Fig. 5b). Compared to the surface of MgA/MAO, the surface of MgA/MAO/SA-Van was more hydrophobic due to the presence of SA and Van (Fig. 5b vs Fig. 5c). After MgA/MAO/SA-Van was cross-linked by CaCl2, the water contact angle changed from 48.3° to 22.4° (Fig. 5c and d). The above mentioned results indicated that the hydrophilicity/hydrophobicity properties of the modified MgA could be tuned by different coatings.
3.2. Evaluation of anticorrosion property of the modified MgA substrate The rapid corrosion of MgA in physiological environment hampers its clinical application. Therefore, the anticorrosion effect of the coating on MgA implant materials surface is of utmost importance to their practical application [32]. Corrosion behavior of different MgA samples was studied by electrochemical corrosion tests. The potentiodynamic polarization curves are shown in Fig. 6. The relevant parameters including the icorr and Ecorr are listed in Table 2. Compared to original MgA (Ecorr = −1.595 V), Ecorr value of the MgA/MAO, MgA/MAO/SAVan and MgA/MAO/cross-linked SA-Van increased by 11, 1209 and 1281 mV, respectively. Compared to original MgA, the corresponding icorr values of MgA/MAO, MgA/MAO/SA-Van and MgA/MAO/crosslinked SA-Van samples decreased by about 2–4 orders of magnitude (Table 2). An excellent anticorrosion performance is largely dependent on a higher positive corrosion potential or a lower polarization current [33,34]. The above mentioned results indicated that all the modified MgA exhibited excellent anticorrosion performance.
Fig. 6. Potentiodynamic polarization curves of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van. Table 2 Ecorr and icorr of different MgA samples by potentiodynamic polarization test. Curves
Ecorr (V)
icorr (A/cm2)
MgA MgA/MAO MgA/MAO/SA-Van MgA /MAO/ cross-linked SA-Van
−1.595 −1.544 −0.386
6.25 × 10−4 2.27 × 10−6 1.68 × 10−8
−0.314
3.34 × 10−7
Table 1 . Chemical element compositions of different MgA samples obtained from XPS. Samples
Mg (at%)
C (at%)
O (at%)
Si (at%)
N (at%)
Na (at%)
Cl (at%)
Ca (at%)
MgA MgA/MAO MgA/MAO/SA-Van MgA /MAO/ cross-linked SA-Van
46.2 40.3 11.0 5.2
23.0 19.8 58.3 59.0
30.8 34.0 23.0 30.0
/ 5.9 1.8 0.3
/ / 4.0 2.3
/ / 1.1 0.2
/ / 0.8 0.6
/ / / 2.4
4
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Fig. 7. Van release behavior of (a) MgA/MAO/SA-Van and (b) MgA/MAO/ cross-linked SA-Van substrates in PBS buffer.
Fig. 8. SEM images of adhered platelets on (a) MgA, (b) MgA/MAO, (c) MgA/ MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
3.3. Evaluation of the Van release behavior for MgA/MAO/SA-Van and MgA/MAO/Cross-linked SA-Van substrates
significantly inhibit platelet adhesion and improve hemocompatibility. Fig. 9A shows that the hemolysis ratios of MgA, MgA/MAO, MgA/ MAO/SA-Van and MgA/MAO/cross-linked SA-Van are 1.30, 0.93, 0.96 and 0.60%, respectively. The hemolysis ratio of MgA/MAO/crosslinked SA-Van is superior to that of other samples. This indicates that the hemolysis ratio of the modified MgA and the original MgA specifies the requirement of the medical standard. When the hemolysis ratio of the samples is less than 5%, the damage to the red blood cells is very light, and hemolysis reaction does not occur. Fig. 9B shows the dynamic coagulation time of different MgA samples. Clearly, the absorbance decreases gradually with time. When the contact time with blood is the same, the absorbance value of MgA/ MAO/cross-linked SA-Van is higher than that of other samples and the curve of MgA/MAO/cross-linked SA-Van shows the slowest decline trend, indicating that the coagulation process of MgA/MAO/crosslinked SA-Van is the slowest.
Furthermore, the Van release behavior of MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van substrates was investigated. Fig. 7 shows that MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van substrates indeed exhibit similar controlled release trend. For the first 25 min, the Van gets released very quickly from two substrates. After 50 min, the release of Van from two substrates enters a plateau phase. Drug loading amounts of MgA/MAO/SA-Van and MgA/MAO/crosslinked SA-Van substrates are 0.68 and 0.36 mg per square centimeter sample, respectively. During the cross-linking process, the loaded Van released into the solution (about 0.32 mg per square centimeter sample), which can be calculated from data presented in Fig. 7.
3.4. Evaluation of the hemocompatibility of the modified MgA substrates 3.5. Antibacterial properties of the modified MgA substrates
Platelet adhesion onto the surfaces of biomaterials triggers the coagulation of blood and thrombus formation [35]. Thus, the property of anti-platelets adhesion is usually used to evaluate the hemocompatibility of biomaterials [36]. In the current study, the morphologies and numbers of the adhered platelets on surfaces of original MgA, MgA/MAO, MgA/MAO/SA-Van and MgA/MAO/cross-linked SA-Van were measured by using SEM images. Fig. 8a exhibits the presence of numerous platelets adhered dendritically on the surface of original MgA. The amounts of platelets adhered on the surface of MgA/MAO (Fig. 8b) decrease obviously compared to that of original MgA. A small amount of platelets adhered on the surface of MgA/MAO/SA-Van, exhibiting good anti-platelet adhesion properties (Fig. 8c). The MgA/ MAO/cross-linked SA-Van showed the best anti-platelets adhesion property, because the adhered platelets could hardly be observed (Fig. 8d). Furthermore, it has been reported that hydrogel coating on substrate surface can enhance anti-platelets adhesion property, and the anti-platelets adhesion ability of hydrogel is affected by the zeta potential, serum proteins adsorption, chemical component and hydrophilicity of the hydrogel [37]. However, success in these fields still remains rare. Chung et al. [38] reported that the synthesized SA-hyaluronic acid hydrogels showed similar hemocompatibility with that of medical-grade polyurethane. Joseph et al. [39] reported that the unmodified polyethylene terephthalate presented thrombogenic behavior, and improved anti-platelets adhesion property after being coated with alginate dialdehyde cross-linked gelatin hydrogel. The Van-loaded SA hydrogel coating reported herein provides an example that can
Apart from the anticorrosion performance and hemocompatibility, implant-related infection is also the major reason for the failure of implant in clinical applications, accounting for high medical cost [10–12]. In this study, the inhibition zone method was selected to estimate the antibacterial activity of the modified MgA substrates against E. coli and S. aureus. The photographs of inhibition zone of original MgA and modified MgA samples are shown in Fig. 10. The original MgA (Fig. 10a) and MgA/MAO substrates (Fig. 10b) have almost no inhibition zone area to either E. coli or S. aureus, which indicates that the original MgA and MgA/MAO samples show little antibacterial activity toward either E. coli or S. aureus. In contrast, MgA/MAO/SA-Van (Fig. 10c) and MgA/MAO/cross-linked SA-Van (Fig. 10d) substrates exhibited clear inhibition zone against S. aureus. The results indicated that the Van released from the SA hydrogel coatings performed better bactericidal effect against S. aureus than that against E. coli. Haldar et al. reported that Van and its derivatives are known to be highly active against various gram-positive bacteria [40]. Actually, in the clinical application, most of the implant-related infections are caused by S. aureus [41]. Therefore, we believe that the approach presented herein is helpful to solve implant-related infections. 4. Conclusions In this study, a Van-loaded SA hydrogel coating was developed on MAO-treated MgA surfaces. The SEM and XPS results confirmed that 5
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Fig. 9. (A) Hemolysis ratio and (B) dynamic coagulation time of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SA-Van and (d) MgA/MAO/cross-linked SA-Van.
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Fig. 10. Inhibition zone photos of (a) MgA, (b) MgA/MAO, (c) MgA/MAO/SAVan, (d) MgA/MAO/cross-linked SA-Van for E. coli and S. aureus, respectively.
Van-loaded SA hydrogel coatings were prepared on the MgA/MAO surface. Furthermore, SEM measurement illustrated that the thickness of the composite coating increased to 25 μm after the hydrogel deposition. The icorr of the MgA/MAO/cross-linked SA-Van significantly decreased approximately by three orders of magnitude compared to that of original MgA sample, exhibiting higher potential for improving anticorrosive property of MgA. More importantly, the MgA/MAO/ cross-linked SA-Van were capable of improving antibacterial properties due to the introduction of Van antibacterial agent in vitro, especially for S. aureus. Moreover, the fabricated Van-loaded SA hydrogel coating showed anti-platelets adhesion, superior hemolysis ratio, and slower coagulation process, indicating excellent hemocompatibility. Overall, the developed platform herein may pave a way toward a versatile, promising strategy for improving the anticorrosive and biological performance of biodegradable MgA in biomedical field.
Acknowledgments This work is supported by the National Natural Science Foundation of China (Nos. 21773149, 51303218), State Key Project of Research and Development (No. 2016YFC1100300), the Key Research and Development Project of Shaanxi Province of China (No. 2018GY-117), Fundamental Research Funds for the Central Universities (2018CDXYSW0023), Chongqing Research Program of Technological Innovation and Application Demonstration (cstc2018jscx-msybX0299) and Innovation Team in University of Chongqing Municipal Government (CXTDX201601002) .
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