Electrophoretic Deposition of Gentamicin-loaded Silk Fibroin Coatings

Stomatol., Union Hospital, Tongji Medical College, Huazhong University of ... to improve the biological functionality of porous structures made of med...
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Electrophoretic Deposition of Gentamicin-Loaded Silk Fibroin Coatings on 3D-Printed Porous Cobalt−Chromium−Molybdenum Bone Substitutes to Prevent Orthopedic Implant Infections Changjun Han,†,‡ Yao Yao,†,§ Xian Cheng,†,§,∥ Jiaxin Luo,§ Pu Luo,§ Qian Wang,‡ Fang Yang,∥ Qingsong Wei,*,‡ and Zhen Zhang*,§ ‡

State Key Lab of Materials Processing and Die and Mould Technology, Huazhong University of Science and Technology, Wuhan, 430074, China § Dept. Stomatol., Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China ∥ Department of Biomaterials, Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands S Supporting Information *

ABSTRACT: In addition to customizing shapes of metal bone substitutes for patients, the 3D printing technique can reduce the modulus of the substitutes through the design and manufacture of interconnected porous structures, achieving the modulus match between substitute and surrounding bone to improve implant longevity. However, the porous bone substitutes take more risks of postoperative infection due to its much larger surface area compared with the traditional casting solid bone substitute. Here, we prepared of gentamicin-loaded silk fibroin coatings on 3D-printed porous cobalt−chromium− molybdenum (CoCrMo) bone substitutes via electrophoretic deposition technique. Through optimization, relatively intact, continuous, homogeneous, and conformal coatings with a thickness of 2.30 ± 0.58 μm were deposited around the struts with few pore blocked. The porous metal structures exhibited no loss in mechanical properties after the anode galvanic corrosion in EPD process. The initial osteoblastic response on coatings was better than that on metal surface, including cell spreading, proliferation and cytotoxicity. Antibacterial efficacy experiments showed that the coatings had an antibacterial effect on both adherent and planktonic bacteria within 1 week. These results suggested that the beneficial properties of anode electrophoretic deposited silk fibroin coatings could be exploited to improve the biological functionality of porous structures made of medical metals. major reasons for the long-term loosening of bone substitute.7 The 3D printing is capable of reducing the modulus of metal bone substitutes through the design and manufacture of interconnected porous structures, achieving the modulus match between metal substitute and surrounding bone to improve implant longevity.6,8−10 The porous bone substitute is easier to obtain postoperative infection due to its much larger surface area compared to the conditional casting solid bone substitute.11 The systemic antibiotics are often used for a few days after orthopedic surgery to prevent potential infection, but they show limited benefits and considerable side effects.12 Thus, it is greatly significant to locally deliver the antibiotics, such as gentamicin, by constructing coating onto the porous bone substitute for the prevention of orthopedic implant infections.13 A variety of strategies to coat the biomaterial surfaces have been developed, such as sol−gels, electro-spraying, and electrophoretic deposition (EPD).14 Among these coating strategies, EPD technique

1. INTRODUCTION The reconstruction of bone defect has remained an important clinical issue as many people suffer from bone diseases caused by trauma, infection, arthritis, tumors, osteonecrosis, osteoporosis, metabolic bone diseases, and other diseases.1 Autologous bone grafting represents the gold standard in treating bone defect, but significant donor-site morbidity and volume restrictions limit its widespread application.2 Some metal alloys, such as cobalt−chromium−molybdenum (CoCrMo), are now widely being used as bone substitutes in the clinic due to their high strength, ductility, and corrosion resistance.3 These metal alloys are very difficult and costly to be manufactured geometrically, similar to the patient-specific defect of bone tissues by using conventional manufacturing methods such as casting, forging, and machining.4 The metal 3D printing technique, such as selective laser melting (SLM), provides the potential to achieve a great degree of design and manufacturing flexibility and efficiency, which could customize these irregular and complex shapes for patients.5 Moreover, the modulus of solid metal bone substitutes made by traditional fabricating techniques is much higher than that of human bone.6 It results in the stress shielding effect, which is believed to be one of the © XXXX American Chemical Society

Received: July 29, 2017 Revised: September 26, 2017 Published: October 3, 2017 A

DOI: 10.1021/acs.biomac.7b01091 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. (A) Design of BCC unit cell (A1) and porous CoCrMo substrate (A2). (B) Schematic illustration of 3D printing of porous CoCrMo substrates by SLM technique. (C) Schematic representation of the possible mechanism of EPD of SFGM. (C1) Pure silk fibroin could be deposited and form a hydrogel coating at positive electrodes due to the screening of negative surface charge of the molecule and the local conformal changes from the random-coil to the helical state on anode. (C2) Gentamicin may form a polyelectrolyte complex with fibroin at first, and then deposited with silk fibroin together at positive electrode due to the anode EPD capability of silk fibroin itself. (D1) Schematic illustration of constructing SFGM coatings on the porous CoCrMo substrates via EPD technique at optimized parameters. (D2) Schematic illustration that hydrogel may turn into fragmentary and discontinuous coatings on struts at too low voltage or for too short time. (D3) Schematic illustration that hydrogel may turn into film contacted with more than one strut to block the pores at excessive voltage or for over a long time.

anode in aqueous environments under an electric field,20 indicating a new kind of EPD basic material. Differing from the conventional EPD basic material, such as positively charged chitosan, the deposition of negatively charged silk fibroin occurs at the positive electrode, and it is known that there is always some extent of galvanic corrosion on anode metal surface due to the galvanic action.21,22 However, the potential risk and worry about uncertain effects of anode galvanic corrosion during EPD on mechanical property of anode metals have not been investigated so far. In this study the gentamicin-loaded silk fibroin coatings were constructed on 3D-printed porous CoCrMo bone substitutes by EPD technique. The crucial EPD parameters were optimized, and the topography of coated porous substrates was observed. The mechanical properties of porous CoCrMo

is a promising method, with the advantages of short processing time, simple equipment requirements, easy control of parameters, and a wide range of possible coating materials.15 Moreover, it is a liquid-based method, and the liquid could flow into the inside of porous structures, indicating it is quite suitable to construct the coatings on porous structures. Silk fibroin, a low-cost natural protein extracted from silkworm cocoons, possesses the self-assembling ability, flexible preparation process, nonimmunogenicity, good biocompatibility and mechanical property.16 It is an FDA (U.S. Food and Drug Administration)-approved polymer that has been widely used in the clinic, such as sutures and drug delivery systems.17 In recent years, many studied have been reported concerning the use of silk proteins in the field of biomaterials and tissue engineering.18,19 It has been reported to form e-hydrogel on the B

DOI: 10.1021/acs.biomac.7b01091 Biomacromolecules XXXX, XXX, XXX−XXX

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2.5. Topography of Coatings. The topography of substrates with and without coatings was inspected by digital camera, fluorescence microscopy (TE-2000, Nikon, Japan), field emission-scanning electron microscopy (SEM; JEOL-JSM7600F, Akishima, Japan), and atomic force microscopy (AFM; SPM9700, Shimadzu, Japan). For fluorescence images, both groups were stained for 15 min with 2-(4amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Invitrogen, Basel, Switzerland), a dye with a relatively strong blue spontaneous fluorescence. This dye could nonspecifically bind with the silk protein but could not bind with metal surface (comparison between Figure 4A1,A2). After dyeing, both coated and uncoated porous substrates were immersed and washed with Milli-Q water for 5 min, and then subjected to five cycles of washing step to remove any unbinding dyes. For measuring the proportion of blocked pores, random 25 pores per sample were recorded continuously, and four samples per group were analyzed. A microcomputer tomography (μ-CT) scanner (diondo d2, Germany) was used to scan the coated substrate at 90 kV voltage and 140 μA current. Two-dimensional (2D) projection image data was collected and the 3D CT volume data was reconstructed from the acquired image series with the diControl CT software (diondo, Germany). During the acquisition, samples were rotated over 360° in steps of 0.2°. After each rotation, 1800 images were acquired and reconstructed into cross-sectional images with the diControl CT software package. This data set was further analyzed and visualized using commercially available image analysis software VGStudio Max 2.2 (Volume Graphics GmbH, Heidelberg, Germany). For measuring the thickness of coating, six cross section images including three random internal struts and three random surface-exposed struts were analyzed, and 25 random positions on the coatings in each cross section image were chosen. 2.6. Phase Identification of Porous Metal Surface Before and After EPD Process. Phase identification of metal surface before and after EPD was performed by X-ray diffraction (XRD) on an XRD7000S instrument (Shimadzu, Japan) with a Cu tube at 40 kV and 30 mA. The scattering angular (2θ) varied from 30° to 90° using a continuous scan rate at 2° min−1. In order to expose the metal surface after EPD process, the hydrogel deposited on the metal surface was immediately and thoroughly washed after deposition and before drying procedure. 2.7. Mechanical Characterization of Porous Substrate Before and After EPD Process. To determine macroscopic mechanical property change of the porous substrates, the axial compression testing was carried out using an AG-IC100 KN Electronic Universal Testing Machine (SHIMADZU, Japan), equipped with a 100 kN load cell, at a constant loading rate of 0.5 mm/min. All samples were loaded to failure along the axial direction. The stress strain data for each tested substrate was calculated from the compressive load versus displacement data obtained from the testing with the measured dimension and gauge length. The elastic modulus and compressive strength were determined from stress strain curves. The microscopic fracture morphologies of the substrates were observed using SEM (JSM-7600F, JEOL, Japan). Hydraulic test frame (EHF-UV100k2-040-1A, Shimadzu, Japan) with a dynamic 100 kN load cell was used for compression-compression fatigue testing. A constant loading frequency (10 Hz, sinusoidal wave shape) and a constant load ratio, R = 0.185, were used. The 0.5σp (σp is referred to the compressive strength) was chosen. Three samples per group were tested. 2.8. Initial Cell Response. MC3T3-E1 (ATCC, USA), an osteoblast precursor cell line derived from Mus musculus (mouse) calvaria, is the most commonly used in vitro osteoblast model.24 Cells were cultured in alpha modified essential medium (HyClone, U.S.A.) supplemented with 10% fetal bovine serum (Gibco, U.S.A.) at 37 °C in a humidified 5% CO2 atmosphere. Coated and uncoated CoCrMo substrates were steam sterilized, placed in 24-well tissue culture plates, and then seeded at a density of 20000 cells·cm−2. After 1, 3, and 7 days of culture, three samples were fixed with 3.7% paraformaldehyde for cytoskeleton observation. Filamentous actins (Factins) were stained with rhodamine phalloidin (R-415 kit, Molecular

structures before and after EPD process were measured. The biocompatibility and antibacterial property of porous CoCrMo substrates with and without coatings were investigated.

2. MATERIALS AND METHODS 2.1. Materials. The commercial CoCrMo gas-atomized spherical powder (Tiger International BioMetals Co., Ltd., China) with a nearly spherical shape was used (Figure S1). The particle size distribution was between 1 and 28 μm, and the chemical compositions of the powder include 62.7% Co, 29.6 wt % Cr, 6.6 wt % Mo, 0.82 wt % Si and 0.3 wt % C. Silk fibroin aqueous solution was prepared according to previous reports.23 Bombyx mori cocoons were boiled for 30 min in an aqueous solution of 0.02 M sodium carbonate, and then rinsed with Milli-Q water. After drying, the extracted silk fibroin was dissolved in 9.3 M lithium bromide solution at 60 °C for 4 h. Then, the fibroin solution was dialyzed against Milli-Q water using a cellulose dialysis membrane (MW = 3500, Biosharp, U.S.A.) for 72 h. Impurities were removed by centrifugation at 5000 rpm for 1h at 4 °C. The final concentration of silk fibroin aqueous solution was approximately 40 mg·mL−1, which was determined by weighing the remaining solid after drying. Finally, the silk fibroin aqueous solution was diluted into 20 mg·mL−1, and then 1 mg·mL−1 gentamicin (G1914, Sigma, U.S.A.) was added into the silk fibroin solution with magnetic stirring of 100 rpm for 1 h to obtain a transparent silk fibroin/gentamicin (SFGM) aqueous solution (Figure S2). 2.2. 3D-Printed Porous CoCrMo Substrates by SLM Technique. As a model of 3D-printed porous bone substitutes, the body centered cubic (BCC) unit cell was designed and used to construct the porous metal substrates (Figure 1A1). It was referred to as octahedron, containing a node located at the center of a cube, from which all the struts radiate out to the corners of the cube. The cell size was 1250 μm and strut size was 200 μm. The cylindrical porous CoCrMo substrates (diameter of 10 mm; height of 4 mm; porosity of 86%) were generated through arraying the BCC unit cells along horizontal and perpendicular planes using the Unigraphics NX8.0 software (Figure 1A2), and then produced by SLM technique (Figure 1B). The HRPM-II SLM machine, developed by Rapid Manufacturing Center in Huazhong University of Science and Technology, was adopted to manufacture the porous CoCrMo substrates. The machine used a fiber laser (redPOWER R4, SPI Lasers, U.K.) with the maximum power of 400 W. The wavelength and laser beam diameter of the laser source were respectively 1064 nm and 100 μm, respectively. The processing was performed in an atmosphere filled with high purity argon in the building chamber. Based on the preliminary work on the process, all the porous substrates were successfully manufactured using the optimized parameters with laser power of 220 W, scan line spacing of 70 μm, scanning speed of 300 mm/s and layer thickness of 20 μm. 2.3. Fabricating SFGM Coatings on Porous Substrates via EPD Technique. In the EPD process, the porous metal substrate was immersed in the SFGM solution. A parallel platinum plate was used as a cathode (Figure 1C). The distance between the positive and negative electrodes was 50 mm. EPD was carried out by connecting both electrodes to a direct current power supply (Model 6614C, Agilent Technologies) with a different period of time from 30 s to 4 min at the same voltage of 20 V, and different voltage from 5 to 80 V for the same time period of 1 min. After EPD, the substrates were air-dried overnight at room temperature, and then cross-linked by water vapor annealing in a vacuum desiccator overnight at room temperature to induce the β-sheet formation of silk fibroin and make silk fibroin insoluble, finally followed by air drying at room temperature again. 2.4. Structural Properties of SF, GM, and SFGM. The measurement of zeta potential of pure gentamicin, pure silk fibroin, and SFGM in aqueous solution was performed by photon correlation spectroscopy (Zetasizer 3000 Malvern Instruments, U.K.). The chemical groups of coatings were determined by Fourier transform infrared spectroscopy (ATR-FTIR; VERTEX 70, Bruker, Germany) at room temperature. C

DOI: 10.1021/acs.biomac.7b01091 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 2. (A) Zeta potential of pure gentamicin, pure silk fibroin, and SFGM in aqueous solution. (B) ATR-FTIR spectra of pure gentamicin powder, pure silk fibroin EPD coatings, and SFGM EPD coatings. Probes, Invitrogen, U.S.A.) for 20 min at room temperature. The nuclei were stained with DAPI for 15 min at room temperature. Additionally, live cells on two groups of porous structures were assessed after being stained with calcein AM (PI; Sigma, U.S.A.) for 20 min at 37 °C. Fluorescence images were obtained using a Nikon TE2000 inverted microscope. The total cell number was quantified at specific time intervals (1, 3, and 7 days) using a cell counting kit (CCK-8; Dojindo, Japan). The result of CCK-8 was tested spectrophotometrically according to the manufacturer instructions. Three samples per group were tested. After 3 days of incubation, Lactate Dehydrogenase (LDH) Assay Kit (Beyotime, China) was used for determination of cytotoxicity by measuring the LDH activity released from damaged cells. The results from the LDH assay were tested spectrophotometrically according to the manufacturer instructions. Three samples per group were tested. The coated and uncoated porous substrates were soaked in 2 mL of ultrapure water and then incubated at 37 °C and gently shaken at 90 rpm. After 3 days, 1 mL of supernatant was collected and the concentration of Co and Cr ions in the supernatant was measured by inductively coupled plasma mass spectrometry. Three samples per group were tested. 2.9. Inhibition Zone. Staphylococcus aureus (ATCC, U.S.A.) was cultured in Muller−Hinton broth (MHB), and then diluted to 1.5 × 108 CFU/mL suspension. A reported disk diffusion method25,26 has been used to estimate the bioactivity of gentamicin loaded in our coated substrates. After dispersion, 100 μL suspension was inoculated by spreading the bacteria suspension evenly over the entire surface of a Muller−Hinton agar plate with a sterile cotton swab, followed by placing the sterile coated and uncoated porous CoCrMo substrates in the dry state. The agar was incubated 37 °C for 7 days, and the inhibition zone was measured after 24 h, 2, 3, 5, and 7 d. The results were listed as the diameter of the inhibition zone. Three samples per group were tested. 2.10. In Vitro Sustained Infection Test. Staphylococcus aureus were cultured by MHB culture solution, then diluted to 106 CFU/mL suspension. Sterile-coated and uncoated porous substrates were placed in 24-well plates, respectively, and 1 mL suspension with 106 CFU/ml live bacteria was added per well. Then the 24-well plates were incubated at 37 °C and gently shaken at 90 rpm. Every 24 h, the medium was replaced with equal amount of fresh MHB with 106 CFU/mL live bacteria. After 1, 2, 3, 5, and 7 days, we transferred medium and substrates into two new in 24-well plates, respectively. For planktonic bacteria testing, 50 μL of WST solution from a Microbial Viability Assay Kit-WST (Dojindo, Japan) was mixed with the medium per well. For adherent bacteria testing, 50 μL of WST solution and 950 μL of MHB were added into the substrate per well. After an incubation of 2 h, 100 μL of supernatant per well were taken

out and tested spectrophotometrically (λ = 450 nm). Three samples per group were tested. The live adherent bacteria on different surfaces were stained by Bacstain-CTC Rapid Staining Kit (Dojindo, Japan) and then observed by Nikon TE-2000 inverted microscope. 2.11. Statistical Analysis. Statistical was performed using SPSS v.20.0, and data were presented as mean ± standard deviation (SD). Student’s t test was used to determine statistical significance, and statistical significance was considered at p < 0.05

3. RESULTS 3.1. Structural Properties of SF, GM, and SFGM. The silk fibroin was negatively charged with zeta potential about −6.9 mV in aqueous solution. On the contrary, the gentamicin was positively charged with zeta potential about +0.6 mV. When the gentamicin was blended into silk fibroin solution, the zeta potential of the mixed solution decreased to −5.6 mV (Figure 2A). ATR-FTIR spectra showed that pure silk fibroin and SFGM coating had an identical absorbance peak of amide I at 1635 cm−1, amide II at 1516 cm−1 and amide III at 1234 cm−1. Pure gentamicin and SFGM coatings exhibited an identical absorbance peak of C−N stretching at 1107 cm−1, and C−O stretching at 1025 cm−1. 3.2. Optimization of Parameter of EPD Process. Due to our symmetrical structure design, the light from fluorescent microscope could penetrate the pores of the substrate if the pores were not blocked by SFGM polymer. At 30 s, almost no blocked pores were observed, but the coatings with blue fluorescence seemed fragmentary (Figure 3A2,A7). As time increased from 30 s to 1 min, the blue fluorescent coatings became much more integral and continuous, still with few pores blocked (Figure 3A3,A8). At 2 min, the majority of the pores become opaque, and the number of blocked pores increased sharply (Figure 3A4, A9, and A11). When the time increased to 4 min, no light could penetrate the pores, indicating that almost all the pores were blocked (Figure 3A5, A10, and A11). The voltage exhibited very similar effects to time. Below 20 V, the coatings seemed fragmentary (Figure 3B2,B7). At 20 V, the struts of the porous structures were covered with relatively integral and continuous SFGM coatings with few pores blocked (Figure 3B3,B8). Above 20 V, the number of pores blocked increased sharply (Figure 3B4−B5 and B9−B10). D

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Figure 3. Optimization of key EPD parameter. (A) Time: (A1−A5) Digital photographs; (A6−A10) Representative in situ merge images of fluorescent and optical microscope; (A11) The proportions of blocked pores. (B) Voltage: (B1−B5) Digital photographs; (B6−B10) Representative in situ merge images of fluorescent and optical microscope; (B11) Proportions of blocked pores.

3.3. Topography of Coatings. The fluorescent and SEM image exhibited that relatively continuous, intact, and conformal SFGM coatings were deposited around the struts of the porous CoCrMo substrate (Figure 4A,B). The AFM image showed that polymer coatings made the surface smoother than the metal substrate (Figure 4C). The μ-CT 3D reconstruction of the whole coated porous structure was shown in Figure 5A. The metal and polymer could be distinguished by the gray level in the reconstructed images, which is related to the different density of the materials.27 High-density CoCrMo metal exhibited light gray, while the SFGM polymer with much lower density was

observed in dark gray. The pores with no density showed black. The representative longitudinal and cross sections presented that few pores had been blocked with the dark gray polymer (Figure 5A2−A5). The representative high magnification cross section image of one coated strut showed relatively homogeneous, continuous, intact and dark gray polymer coating was deposited around the metal strut surface in light gray with an average thickness of 2.30 ± 0.58 μm (Figure 5A6,A7 and Figure 6B). The thickness of coatings on surfaceexposed struts showed no significant difference compared with that on the internal struts (Figure 6A). The high magnification 3D reconstruction of the representative two adjacent unit cells E

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Figure 4. Topography of coated and uncoated porous CoCrMo substrates. (A) Representative in situ merge images of fluorescent and optical microscope. (B) Representative SEM images. (C) Representative AFM images. The red arrow indicates the SFGM coating and the yellow arrow indicates some unmelted CoCrMo powder particles attached to the strut during SLM.

8A1,A2) and CCK-8 cell proliferation test (Figure 8C1) presented that there was no significant difference in cell number between coated and uncoated substrates. However, in the fluorescent images of cytoskeleton (Figure 8B1), osteoblastic cells on SFGM coatings exhibited excellent spreading and extension with many pronounced and elongated filopodia pseudopodia (Figure 8B7, yellow arrowhead) and cell junctions from the cell edges (Figure 8B7, white arrowhead). The cells on the metal surface seemed more rounded in shape and spread with fewer microextensions (Figure 8B8). In day 3, cell number increased but showed no significant difference in both groups (Figure 8A3,A4 and Figure 8C1), while the cell spreading area on SFGM coatings was obviously larger than the metal surface (Figure 8B3,B4). Besides, the cytotoxicity (Figure 8C2), Co and Cr ion release (Figure 8C3,C4) of coated porous substrates were observed significantly lower compared to the uncoated ones. After day 7, fluorescent images and CCK-8 results exhibited obviously better cell proliferation on SFGM coatings (Figure 8A5,A6,C1). The entire surface of the coated substrate was covered with flat and well-spread cells with numerous microextensions and well-developed connections of cellular protrusions (Figure 8B5). On the contrary, less cell spreading could be seen on the CoCrMo substrate, and the cells on metal surfaces even had not formed a complete confluent layer (Figure 8B6). 3.6. Antibacterial Efficacy of Coatings. The inhibition zone results showed that its average diameter did not decrease significantly within 1 week. (Figure 9A). In vitro continuous infection test or the WST-8 test indicated there were almost no live adherent (Figure 9B1) and planktonic bacteria (Figure 9B2) surviving in the group of coated porous structures within 5 days. At 7 day, some live adherent and planktonic bacteria appeared, but their number was still far less than that in uncoated CoCrMo groups. The number of live adhered bacteria on the metal substrates without SFGM coatings increased with the increasing of time within 1 week, while the

also showed the relatively continuous, intact, and conformal topography of the SFGM coatings (gray) around the struts of porous CoCrMo substrate (green) (Figure 5B). 3.4. Mechanical Properties of Porous Substrates Before and After EPD Process. The anode EPD process would exist some extent of galvanic corrosion on the anode metal, which is one of the major concern about its potential undesirable effect on the mechanical performance of metal. The XRD tests were conducted to identify corrosion effect through phase variation of surface before and after EPD. The results exhibited that the peaks of face-centered cubic (fcc) γ phase of Co and Cr were dominantly detected at 74.8°, 50.9°, and 43.6°, respectively, and no other obvious peaks appeared on the metal surface before EPD. In contrast, after experiencing the anode corrosion during the EPD, the characteristic peaks of γ phase of metal surface slightly shifted to 74.5°, 50.6° and 43.4° respectively, indicating the variation of lattice plane distance in the microstructure of CoCrMo surface. More significantly., the small amounts of Co3Mo (74.5°, 46.4°, and 43.4°) and Cr23C6 carbides (82.9°, 74.5°, 63.1°, and 50.6°) phases could be additionally identified on the surface of substrate (Figure 7A). Both the variation of lattice plane distance and appearance of these new phases indicated that there could be some extent of microstructure change of the metal surface due to the anode galvanic corrosion. Macroscopically, the compressive strength and fatigue life of the porous substrate presented no significant change after the anode galvanic corrosion in EPD process, while the elastic modulus slightly increased (Figure 7B). Microscopically, the SEM images of compression fractures exhibited no significant difference before and after corrosion (Figure 7C). At a high magnification of both group were presented a lot of river patterns (yellow arrows) and cleavage steps (red arrows) along with a specific direction (white arrows) that indicated cleavage fractures, which results in the wedge-type cracks at low magnification (blue arrows). 3.5. Initial Cell Response of Coatings. After 24 h of culture, the fluorescent images of live cell staining (Figure F

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Figure 5. μ-CT analysis of coated porous CoCrMo substrate. (A1) 3D reconstruction of the whole coated porous substrate. (A2) Representative longitudinal section of a2 plane (red) in A1. (A3−A5) Cross section at three representative height level of a3, a4, and a5 plane (red) in A2. (A6) High magnification image of the representative area (red) in A4. (A7) High magnification image of the representative area (red) in A6. (B) High magnification image of a 3D reconstruction of the representative two adjacent coated unit cells (blue dotted frame) in A2. (B1) 3D reconstruction for the topography of CoCrMo substrate (green) with SFGM coating (gray). (B2) 3D reconstruction only for the topography of polymer coating (gray) of the upper unit cell in B1. (B3) 3D reconstruction only for the topography of metal substrate (green) of the upper unit cell in B1. The yellow arrows indicate the SFGM coatings and the green arrows indicate the CoCrMo substrates.

SFGM coatings within 5 days of continuous infection (Figure 9C). After 7 days of sustained infection, there was only very little viable bacteria colonization observed on coatings (Figure 9C3). However, bacteria colonization increased constantly on the uncoated metal surface, and completely colonized the metal surface in day 7 (Figure 9C9).

4. DISCUSSION The EPD coating is an attractive and promising method for surface modification of porous metal structures due to its aqueous preparation condition. In the present study, relatively intact, continuous, and homogeneous SFGM coatings were deposited on 3D-printed porous CoCrMo substrates after optimization of crucial EPD parameters. The constructed coating was very thin and showed little effects on the original topography design of the porous structure. The coated porous substrates provided sustained antibacterial properties within 1

Figure 6. (A) Thickness of the coatings around external and internal struts measured by μ-CT. (B) Thickness distribution of the coatings measured by μ-CT.

number of planktonic bacteria was at a high level and showed no obvious variation with time. The fluorescent images exhibited that almost no live bacteria could adhere onto the G

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Figure 7. Mechanical property of porous substrates before and after EPD process. (A) XRD of metal surfaces before and after EPD process. (B) Macroscopic mechanical properties of coated and uncoated porous substrates: (B1) Stress strain curves; (B2) Compressive strength; (B3) Elastic modulus; (B4) Cycles to failure according to fatigue tests. (C) Representative microscopic morphology of compression fractures by SEM. The blue arrows indicating the wreck cracks, the red arrows indicating the cleavage steps, the yellow arrows indicating the river patterns, and the white arrow indicating the specific direction of river patterns and cleavage steps. Asterisk (*) denoting significant differences (p < 0.05).

to become very low near the anode, and silk fibroin has been reported to form electro-hydrogel at positive electrodes due to a screening of the negative surface charge by H+ and local conformal changes from the random-coil to the helical state on anode (Figure 1C1).20 Positively charged pure gentamicin should accumulate on cathode under an electric field. However, in our study the gentamicin was found to be deposited with silk fibroin together at positive electrode instead of negative one. It suggested that polyelectrolyte complex formation might be a critical step in our EPD process. The gentamicin may form polyelectrolyte complex with silk fibroin in the solution at first, and then deposited with silk fibroin together at the positive electrode, which could be driven by the anode EPD capability of silk fibroin itself (Figure 1C2). EPD process is carried out in aqueous environments, and the EPD solution could flow into the inner part of porous structures to construct coatings. The other commonly used coating techniques, for example, plasma spraying, do not allow for creating a continuous and homogeneous coating inside a porous structure,14 but EPD, the technique developed in the study, could solve this problem. The porous substrate was immersed in the SFGM solution, and the SFGM solution

week as well as good biocompatibility, while the anode galvanic corrosion on the metal substrate during EPD was proved to have no obvious adverse effects on the macroscopic mechanical property. The FTIR showed that no new peaks appeared and no obvious peaks shifted. It suggested that no extra chemical reactions and formation of covalent bond occurred between silk fibroin and gentamicin, and their secondary structure remained intact and stable during electrophoretic deposition.28 Silk fibroin, with a pI of 4.2, is negatively charged in aqueous solution.29 When the positively charged gentamicin was blended, the pH of silk fibroin aqueous solution changed from 7.5 to 7.1, and the zeta potential decreased from −6.9 to −5.6 mV. Some negative charges in the biopolymer backbone of silk fibroin should be balanced by the positively charged gentamicin, indicating that the polyelectrolyte complex might be formed by the electrostatic interaction between the anionic groups of silk fibroin and the cationic amino group of gentamicin.30 It is important to note that electrophoresis provides the accumulation of charged particles or polymer macromolecules at the electrode surface, but not necessarily results in the deposit formation.31 The pH would be expected H

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Figure 8. Initial cell response on the coated and uncoated CoCrMo substrates. (A) Representative fluorescent images of live osteoblastic cells (green). (B) Representative fluorescent images of cells labeled with F-actin (red) and nuclei (blue). Yellow arrows indicating filopodia and pseudopodia, and white arrows indicating cell junctions. (C1) Cell number analyzed by the CCK-8 assay. (C2) Cytotoxicity measured by LDH assay. (C3, C4) Co and Cr ion release measured by ICP-OES. Asterisk (*) denoting significant differences (p < 0.05).

might contact and merge with each other due to its high stickiness.20 After the air dying, the merged hydrogel may turn into a film bridging across different struts instead of a film-like coating covering only single strut, making it possible to block the pores of porous structures (Figure 1D3). Under the proper EPD condition (20 V and 1 min), most of SFGM may be deposited and form hydrogel with a moderate thickness on one strut surface, and then turn into a relatively intact film-like coating around that strut surface after drying procedure (Figure 1D1). Digital, fluorescence, SEM, and AFM images, as well as μCT, as noninvasive tools, showed that relatively homogeneous, continuous, intact, and conformal SFGM coatings were deposited on the surface of the external and internal struts with very few pores blocked using optimized EPD parameters. Pore interconnectivity is an important factor of architectural bone scaffold determining the biological outcome.34 An excess of blocked pores would limit cell migration and nutritional exchange.35 The thickness of the coatings was 2.30 ± 0.58 μm, which was negligible compared with the average pores size (625.0 ± 54.1 μm) and strut size (254.2 ± 18.1 μm). The 3D printing technique has been employed to fabricate complex

formed hydrogel on the inner and outer struts of the porous structure simultaneously, immediately after the direct current power supply was connected. The relationship between deposit mass, deposition time, and voltage could be qualitatively described with the Hamaker equation, M = μSCEt

(1)

The equation predicts a linear increase in deposit mass with increasing electric field and deposition time, where M is the mass of a deposit obtained during deposition time t on the electrode area S, and C and μ are concentration of polymer and its electrophoretic mobility, respectively.32 To optimize of these two key EPD parameters of time and electric field, we used digital and fluorescent microscopy images, as a simple, low-cost, fast and effective method to preliminarily evaluate the integrity of coatings and interconnectivity of pores. Our results indicated that for too short deposition time or at a too low voltage, the hydrogel deposited on the strut surface might be too thin to form intact coatings, due to the volume shrinkage and crack formation during air drying of silk fibroin hydrogel33 (Figure 1D2). On the contrary, the deposited hydrogel may become quite thick with too long deposition time or too high voltage. Some of the hydrogels deposited on the adjacent strut surfaces I

DOI: 10.1021/acs.biomac.7b01091 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

Figure 9. Antibacterial efficacy of coated and uncoated CoCrMo substrates. (A1) Diameter of inhibition zone. (A2) Digital photographs of inhibition zone. (B) The number of live adherent and planktonic bacteria in in vitro sustained infection test analyzed by the WST assay. (C) Fluorescent images of live adherent bacteria.

and 3.77 ± 0.34 MPa, respectively (Figure S4). This level of bond strength of EPD coatings would be sufficient to resist the force during press-fit placement in rabbit tibiae.43 Moreover, in this study the majority of the coating was deposited on the internal struts of the porous structure, where the coatings subject no insertion force during the surgery. Initial osteoblastic response to the bone substitutes plays a key role in biocompatibility of the biomaterials, and it is the basis and the early stage of osteogenic function.44 The osteoblastic cells spread better on SFGM coatings with many pronounced and elongated filopodia and pseudopodia. It has been reported that filopodia and connections of cellular protrusions provide the routes for transporting nutrients and exchanging information, which plays a crucial role in cell communication.45 The Co and Cr ions released from the CoCrMo alloy used in our study have been reported to show certain cytotoxic effects against the cells, which may cause the DNA damage46 and oxidative stress reaction.47 Shah et al. found that both Co and Cr are most highly localized at nuclear and perinuclear sites in osteoblasts and described the detrimental influence of cobalt and chromium ions on osteoblasts.48 The cytotoxicity on coating surfaces in our study was less than that on CoCrMo surface, which may result from the significant reduction of the release amount of Co and Cr ions. Both groups had no significant difference in the cell number at first 3 days, but coated substrates exhibited obviously better cell proliferation after a week. It may be caused by the better cell spreading and less cytotoxicity of coated groups that we have discussed above.

porous metal structure imitating the natural porous bone structure with pore size at hundreds of micrometers in many recent studies,5,36,37 and thus, our conformal thin coatings could reserve the individually biomimetic topography design of the porous structure to the extent possible. The elastic modulus, compressive strength, and fatigue life are the main indicators for bearing high loadings, especially for the clinical application of metal bone substitutes.38 It is generally thought that the effect of ultrathin polymer coating on the mechanical properties of metal structure could be negligible.39,40 Nevertheless, in our study the EPD process of SFGM occurred at the positive electrode and porous metal substrate acted as anode. One of the major concern about anode EPD process was that there would always be some extent of galvanic corrosion on the anode metal.41 It may lead to potential undesirable effects on the mechanical performance of the metal.42 XRD results showed that there was some extent of microstructure change of the metal surface due to the anode galvanic corrosion. However, from a macro perspective, neither compressive strength nor fatigue life of the porous metal structures after EPD decreased as suspected, while the elastic modulus was enhanced slightly. The microscopic fracture morphologies of the metal substrates before and after EPD were quite similar. These suggested that the microstructure change resulting from anode galvanic corrosion might be too slight to affect the macroscopic mechanical properties under the relatively mild EPD condition (not very high voltage and short time) applied in our study. The tensile and shear bond strengths of coatings prepared in the study were 3.43 ± 0.38 J

DOI: 10.1021/acs.biomac.7b01091 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules Author Contributions

Gentamicin has been reported to be a widely used antibiotic in orthopedic surgery,30,49 and Staphylococcus aureus have been demonstrated as one of most common pathogenic bacteria in infections associated with surgical implants.50 Gentamicin could bind to ribosome in the bacterial cells and causes production of abnormal proteins, leading to an efficient antibacterial effect on staphylococcus.51 The inhibition zone test showed gentamicin loaded in our coated substrates had the bioactivity on Staphylococcus aureus within 1 week. In in vitro persistent infection test, both adherent and planktonic bacteria were completely inhibited by the coating within 5 days, and the coatings still had significant antibacterial effects within 1 week. Bacteria and host cells compete to adhere, replicate, and colonize on the implant surface at early stage after the surgery,52 and the good antibacterial effect and initial cell response of our SFGM coatings within 1 week would help the host cells to win the match. In the clinic, systemic antibiotics are usually only used for first few days after a major orthopedic surgery. After that, the wound healing process starts and the immune defending system is strong enough to protect against invasion of the bacteria.53 Our SFGM coatings on 3D-printed porous CoCrMo bone substitutes by EPD technique could be used for preventing orthopedic implant infection.

†

These authors contributed equally. 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 The authors are grateful for the support from the National Natural Science Foundation of China (51375189), Fundamental Research Funds for the Central Universities (2015ZDTD028), and the China Scholarship Council (Project No. 201606160095).

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5. CONCLUSION In this study, we constructed gentamicin-loaded silk fibroin coatings on 3D-printed porous CoCrMo bone substitutes by using EPD technique. Through optimization, relatively intact, continuous, homogeneous, and conformal coatings with average thickness of 2.30 ± 0.58 μm were deposited around the struts with few pores blocked. The porous metal substitutes showed no obvious loss in mechanical properties after the EPD process. The initial osteoblastic response on coated substrates was better than that on metal surface, and the SFGM coatings had significant antibacterial effect within 1 week. This work would be beneficial for exploiting the beneficial properties of silk fibroin EPD coatings to improve the biological functionality of porous structures made of medical metals.

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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/acs.biomac.7b01091. SEM and EDS of the CoCrMo spherical powder; digital photograph of different concentrations of gentamicin added into silk fibroin solution; μ-CT analysis of porous CoCrMo substrate; tensile and shear bonding strength of the coatings; cumulative release kinetics of gentamicin by HPLC; cell morphology on cell culture plate after 24 h; inhibition zone of standard gentamicin disk after 24 h (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-13296512995. *E-mail: [email protected]. Tel.: +8613995672381. ORCID

Zhen Zhang: 0000-0003-2228-4078 K

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