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Bioinspired and Biomimetic AgNPs/Gentamicin-Embedded Silk Fibroin Coatings for Robust Antibacterial and Osteogenetic Applications Wenhao Zhou,† Zhaojun Jia,† Pan Xiong,† Jianglong Yan,† Yangyang Li,† Ming Li,‡ Yan Cheng,*,† and Yufeng Zheng†,§

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†

Academy for Advanced Interdisciplinary Studies and §Department of Advanced Materials and Nanotechnology, College of Engineering, Peking University, Beijing 100871, China ‡ China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing 100053, China S Supporting Information *

ABSTRACT: With the progressively increasing demand for orthopedic Ti implants, the balance between two primary complications restricting implant applications is needed to be solved: the lack of bone tissue integration and biomedical deviceassociated infections (BAI), where emergence of multiresistance bacteria make it worse. Notably, a combination of silver nanoparticles (AgNPs) and a kind of antibiotic can synergistically inhibit bacterial growth, where a low concentration of AgNPs has been confirmed to promote the proliferation and osteogenesis of osteoblasts. In this work, we built AgNPs/gentamicin (Gen)embedded silk fibroin (SF)-based biomimetic coatings on orthopedic titanium by a facile dipping−drying circular process and with the assistance of polydopamine (PD). Ag+ was reduced to AgNPs by SF under ultraviolet (UV) irradiation, and then they were detected by transmission electron microscope (TEM) images and UV−visible (UV−vis) analyses. Intriguingly, the addition of Gen highly improved the reduction efficiency of Ag+. The antibacterial efficiency of SF-based coatings was examined by challenging them with pathogenic Staphylococcus aureus (S. aureus) bacteria which produced biofilms, and consequently, we found that low concentration loading, durable release of Ag+ (28 days), and 10-fold improvement of antibacterial efficiency were achieved for our novel AgNPs- and Gen-embeded silk fibroin coatings. In bacteria and a cells cocultured system, AgNPs/Genembedded coatings strongly inhibited adhesion and proliferation of S. aureus, simultaneously improving cell adhesion and growth. To investigate cytocompatibility and osteogenic potential, different coatings were cultured with MC3T3 cells; AgNPs/Genembedded coatings showed generally acceptable biocompatibility (cell adhesion, proliferation, and viability) and accelerated osteoblast maturation (alkaline phosphatase production, matrix secretion, and calcification). Expectantly, this novel biofunctional coating will have promising applications in orthopedic and dental titanium implants thanks to its excellently antibacterial, biocompatible, and osteogenic activities. KEYWORDS: silk fibroin, AgNPs, gentamicin, antibacterial, osteogenic

1. INTRODUCTION Profited from good biocompatibility, corrosion resistance, and wear resistance as well as high strength, pure titanium (Ti) and Ti-based alloys have been used worldwide in orthopedics and dentistry.1,2 Unfortunately, the infection associated with Ti implants is always a severe complication during and after surgery, which can cause delayed healing, implant failure, and repeated surgeries.3−5 To solve the infection problem, various kinds of functional coatings have been developed and proven to be effective to endow implants with self-antibacterial ability, such as antibiotics-loaded coatings,6 inorganic bactericidedoped coatings,7 bioactive antibacterial polymerization,8 and adhesion-resistant coatings.9 Silver-containing coatings have attracted lots of attention to be applied on BAI inhibition owing © 2017 American Chemical Society

to their superior performance including broad antimicrobial spectrum, well-behaved biofilm-inhibited capability, and good stability. Compared to other silver compounds, AgNPs have a higher antibacterial activity, ascribed to both the effects of eluted silver ions and the physical properties of nanoparticles.10 It is considered that AgNPs most likely adhere to the bacterium surface to alter its membrane properties and to inflict damage on DNA by passing through the membrane.11 AgNPs can also release silver ions which are known to exhibit antimicrobial properties via interacting with thiol-containing proteins to Received: May 13, 2017 Accepted: July 21, 2017 Published: July 21, 2017 25830

DOI: 10.1021/acsami.7b06757 ACS Appl. Mater. Interfaces 2017, 9, 25830−25846

Research Article

ACS Applied Materials & Interfaces

Figure 1. (a) Schematic of in situ preparing SF/AgNPs/Gen composite solution and the possible reduction mechanism of Ag+ by SF as well as the interaction between AgNPs and gentamicin. (b) Illustrative diagram of fabrication process for PD-S-Ag/g coatings on titanium substrate. Scheme is not in real scale.

collaborated with Gen to eradicate bacteria. Silk fibroin extracted from Bombyx mori silkworm is usually applied in biomedical coatings owing to its biocompatible and substraterecognized capabilities. An environment friendly and energyefficient synthetic route has been reported to form SF-AgNPs composites involving silk fibroin reducing silver ions while dispersing and stabilizing the produced AgNPs.21 Different from other available reductive polymers, SF is a good option for osteogenic regeneration as well.22−24 The structure of its fibrins is extremely similar to Col I, which is able to build up the bone organic phases. Also, the amorphous connections in the fibroin β-sheet structures tightly imitate the anionic character of noncollagenous proteins, acting as nucleation sites for the crystallization of HA-nanocrystals.25−27 Additionally, the secondary structure of silk fibroin β-sheet crystal endows it with superior mechanical properties, cytocompatibility, and minimal immunogenicity. Furthermore, B. mori silk fibroin films promote steady conglutination with extended filopodia and lamellopodia of human osteoblast cells, accompanying with sustained extensive accumulation of mineralized nodule across the surface even in nonosteogenic medium.28 Benefiting from its promoted osteoblast attachment and well-behaved osseointegration, SF-based coatings are broadly employed in bone tissue engineering. For the first time, a SF-based coating containing AgNPs and Gen was constructed for synergistic antibacterial applications. In order to firmly immobilize SF-based coating on Ti

impair their activities.12 Bacteria can hardly resist silver for the reason that AgNPs likely exert several bactericidal mechanisms in parallel.12 Recently, AgNPs amended with conventional antibiotics showed strong antimicrobial capability, even against multidrugresistant bacteria. The evaluation was applied based on three kinds of antibiotics, polykeptide (tetracycline), β-lactam (penicillin), and aminoglycoside (neomycin), with varying chemical structure and reaction modes. It was observed that the combination of AgNPs with tetracycline or neomycin led to a synergistic inhibitory impact on the growth of multidrugresistant bacteria, while an attempt with penicillin did not show any synergy.13,14 Some mechanism studies15,16 have proved that the combination of AgNPs with antibiotics inhibited the formation of biofilm, which is primarily responsible for antimicrobial resistance and chronic bacterial infections. Others17 pointed out that AgNPs generated hydroxyl radicals to enhance the bactericidal effects. Moreover, a hypothesis has been made that individual bactericidal behaviors of antibiotics and AgNPs were replaced by the formation of AgNPs− antibiotics complexes with stronger antibacterial ability.18 As an aminoglycoside antibiotic, Gen is broadly used to treat many types of bacterial infections and has been proved to show strong synergistic abilities with AgNPs to kill bacteria and inhibit biofilm.19,20 In this article, a biomimetic SF-based coating was established to load antibacterial agents where particulate silver is 25831


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ACS Applied Materials & Interfaces substrates, an assisted polydopamine (PD) film shall be introduced. In the past few years, mussel-inspired dopamine has emerged as an important and versatile building block for modifying biomaterial interfaces. More specifically, it is able to instantly and irreversibly attach to the surface of titanium substrate in alkaline media, resulting in a fixed and thin PD film, allowing the anchorage of rich in amine or sulfhydryl groups.29 Assisted by PD, a rigid PD-S-Ag/g coating can be easily fabricated on Ti. The aims of the present work were (1) to manipulate and characterize AgNPs reduced by SF, (2) to develop and characterize PD-S-Ag/g coatings, where β-sheet secondary structure deeply influences the physical−chemical properties of SF-based coatings, (3) to assess the synergistic antibacterial activities of AgNPs- and Gen-contained coatings according to their antiadhesion, planktonic-killing, and antibiofilm evaluations; (4) to investigate in vitro how osteoblast-like MC3T3 cells react with the AgNPs/Genembedded coatings, in terms of attachment, proliferation, alkaline phosphatase activity, collagen secretion, and ECM mineralization. In this work, biomimetic SF-based coating containing Gen and AgNPs would be expected to show synergistic planktonic-killing and biofilm-inhibition capabilities in vitro and show acceptable biocompatibility and osteogenetic activities.

2.4. Surface Characterization. Surface morphology was obtained by field-emission scanning electron microscopy (FE-SEM, S4800, Hitachi), and the topological structure and surface roughness were obtained by atomic force microscopy (AFM, DimensionICON, Bruker) in contact mode. X-ray photoelectron spectroscopy (XPS) was performed on an AXIS Ultra spectrometer (Kratos Analytical, Manchester, U.K.) with Al Kα excitation radiation (1486.6 eV). UV− vis spectra were detected by a Hitachi U-2910 spectrophotometer. Fourier transform infrared (FTIR, Nicolet, Madison, WI) spectra were collected in transflective mode in the range of 700−4000 cm−1. To characterize the secondary structures of SF-based coatings from the spectra, Peak fit software was used to investigate the amide I region (1595−1705 cm−1) by Fourier self-deconvolution. After area normalization for the deconvoluted amide I spectra, the fraction of the secondary structural units was determined by the single-band relative areas. 2.5. Contact Angle (CA) Measurements. The water contact angle on different specimens was determined by the sessile-drop water method using a SL200B Contact Angle System (KINO, Norcross, GA). DI water was well stayed on the specimen, and measurements were repeated in triplicate at six different positions per substrate type. 2.6. Protein Adsorption. Bovine serum albumin (BSA) acted as a model protein. BSA solution (1 mg/mL, pH 7.4) was transferred gently onto different specimens and coincubated at 37 °C for 2 h. Samples were washed with phosphate-buffered saline (PBS), and the adsorbed proteins were eluted using 2% sodiumdodecyl sulfate (SDS, Sigma) under shaking at 37 °C for 2 h. Quantifiably, a Micro BCA Protein Assay Reagent Kit (Thermo Scientific) was applied to measure absorbance at 570 nm on a microplate reader (Bio-RAD, Hercules, CA). 2.7. Ag+ and Gen Release Measurement. Samples (n = 3) were submersed in phosphate buffer solution (PBS, pH = 7.4) at 37 °C under static conditions. At predetermined times, the leaching medium was collected and freshly added. Analysis was performed by using inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7700×, U.S.). Besides, the total Ag content per sample was determined similarly after dissolving the particles in HNO3 (n = 3). The in vitro release study of Gen was implemented in a plastic tray as follows: samples were placed at the bottom of each well in 24-well plates. A 1 mL amount of phosphate buffer saline (PBS at pH 4.3 and 7.3) was added to each well, and the trays were kept in a humid chamber for incubation at 37 °C. The medium collection was conducted after each set time interval, and then the sample was washed with 1 mL of PBS followed by the addition of 1 mL of fresh PBS. After centrifugation applied on the collected medium, the supernatant was extracted for concentration analysis, before which the extracted supernatant was 20 times diluted. Then 0.05 mL of the diluted samples reacted with 0.75 mL of fluorescamine solution (acetone, 0.15 mg/mL). Also, buffer borate (pH 8.5) was used to dilute the reacted solution to 2 mL followed by 5 min incubation at room temperature. Finally, the complex was analyzed as in the method mentioned before. The experiment was repeated three times. 2.8. Antimicrobial Activity Assays. 2.8.1. Bacteria Culture and Inoculation. Under sterile condition, we incubated cultured S. aureus in Luria−Bertani (LB) broth at 37 °C for 12 h, and then the activated bacteria (106−107 CFU/mL) were cocultured with specimens (sterilized by ultraviolet) for predesigned time periods. All experiments and measurements were carried out in triplicate. 2.8.2. Microbial Viability Assay. A handy WST8-based microbial viability assay (Dojindo, Kumamoto, Japan) was used to investigate the ability of SF-based coatings in inhibiting invasion of bacteria by the established process. In a word, the production of microbial metabolism could react with WST-8 and an electron mediator, resulting in a color change of the LB solution, which could be measured by the colorimetrical method. The antibacterial rates for adhered bacteria (Raa) were thus calculated Raa (%) = B/A × 100, where A and B are average CFU counts of bacteria from sonication solutions for Ti and other specimens. 2.8.3. Characterization of Adherent Bacteria. After fixation with 2.5% (v/v) glutaraldehyde (GA) and dehydration in serial ethanol

2. EXPERIMENTAL SECTION 2.1. Preparation of Silk Fibroin (SF) Solution. Sercin, a hydrophilic gelatinous coating protein, helps bonding of the fibroin fibers constituting the raw B. mori cocoon silk. The removal of sericin and B. mori cocoon silk-dissolving process were implemented as the established procedures.9 Briefly, sercin removal was accomplished by twice treatment with 0.5 wt % NaHCO3 aqueous solution at 100 °C for 30 min, followed by washing with distilled water and drying in the air at room temperature. After degumming, B. mori silk fibers were added into 60 °C 9.3 mol/L LiBr aqueous solution to dissolve. The dialysis of the silk fibroin-LiBr solution against deionized water to get rid of the salt was conducted with a semipermeable membrane (MEMBRA-CEL, 12 000−14 000 MWCO) at room temperature for 72 h. Centrifugation was applied on the dialyzed solution at 6000 r/ min for about 5 min, and then the supernatant was stored at 4 °C. The concentration of the final SF solution was around 10 wt %, and then deionized water was used to dilute to 5 wt %. 2.2. Preparation of SF/AgNPs/Gen Composite Solution. AgNO3 (4−240 mM) and Gen powders (0.4−2.4 g) were added into 2 mL of 5 wt % SF solution to produce a transparent SF/AgNO3/ Gen mixture solution. The final AgNO3 and Gen concentration in the solution was 2−120 mM/L and 0.2−1.2 mg/L, respectively. Afterward, a UV lamp (40 W, from Philips) was used to expose the SF/AgNO3/Gen solution under the ultraviolet, and the solution was cultured at room temperature for 1−5 h to form SF/AgNO3/Gen composite solution. Then SF/AgNO3/Gen composite solution was reserved at 4 °C for further use. 2.3. Building of SF-Based Coatings on Ti. cTi discs (10 mm × 10 mm × 0.5 mm) were mechanically polished up to 2000 grit and rinsed with ultrasonication in acetone, ethanol, and deionized water (DI) in sequence. Then the discs were dried by blowing with purified nitrogen gas to get spotless and dehydrated surfaces for next decoration by polydopamine. Briefly, samples were placed into 2 mg/mL dopamine hydrochloride (Alfa Aesar) with Tris-HCl buffer (10 mM, pH = 8.5; Sigma) for 24 h while constantly vibrating in the dark at 37 °C, and then the excess monomer and particles were isolated by thorough ultrasonication to produce polydopaminedecorated Ti (Ti-PD). PD-S, PD-S-Ag, and PD-S-Ag/g functional coatings were obtained by a simple dipping−drying circular process in SF, SF/AgNPs, and SF/AgNPs/Gen solutions, respectively (Figure 1b). 25832


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Figure 2. (a) UV−vis spectra of the SF/AgNPs and SF/AgNPs/Gen composite solution with different UV irradiation time (1 or 5 h) and SF solution concentration (2 and 5 wt %). (b) TEM images of the SF/AgNPs and SF/AgNPs/Gen composites with different UV irradiation time (1 or 5 h). 2.8.5. ormation of Reactive Oxygen Species (ROS). The production of ROS was assessed using a sensitive 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescent stain (Jiancheng Biotech, Nanjing, China). After coculture of SF-based coatings and bacteria for 1 day, the medium was refreshed by dilute DCFH-DA (nonfluorescent) and incubated for another 2 h at 37 °C, forming 2′,7′dichlorofluorescin (DCF, fluorescent) with a green color. CLSM fluorescence images were collected at 488 (Ex) and 535 (Em) nm in triplicate, and ImageJ software was used for data analysis. 2.8.6. Coculture of S. aureus and MC3T3 Cells. S. aureus was chose as invaded bacteria, which was involved in most orthopedic infections.4 First, MC3T3 cells were sterilely incubated with SF-based coatings for 12 h, and then the culture medium was completely abandoned, and the coatings were rinsed by PBS 3 times. The cells−adhesive coatings were put into new plates containing fresh medium (antibiotic-free α-MEM). Next, 1 mL of S. aureus (1 × 105 CFU/mL) was added into each plate and cultivated for another 3 h to allow bacterial attachment. After gentle wash of the contaminated coatings with α-MEM, aliquots of Gen (100 μg/mL) were added and retained for another 2 h to eradicate the extracellular bacteria. After being incubated for 1-, 3- and 5-days, cell proliferation was quantified using Cell Counting Kits and

(50−100%), the morphologies of anchored bacteria were investigated by SEM observations. Before SEM observing, the dried samples needed to be spluttered with gold particles to increase electrical conductivity. Additionally, S. aureus stayed on SF-based coatings was stained by Live/Dead BacLight Bacterial Viability Kits (Invitrogen) to visualize the living state of bacteria. Briefly, the coatings were rinsed, and 1 mL of stain mixture (6 μM SYTO 9; 30 μM propidium iodide (PI)) was added and kept in the dark for 15 min. The final fluorescent images formed by CLSM observation in XYZ scanning mode. The results were verified by repeatedly observing at more than 5 random regions. 2.8.4. Anti-Biofilm Test. The antibiofilm ability of SF-based coatings was investigated by incubating with bacteria (1 × 108 CFU/mL) for 10 days. The culture medium was refreshed every 3 days. First, the specimens were rinsed by PBS 3 times to remove loose biofilm fragments and then stained by 1% (w/v) crystal violet for 15 min. The specimens were washed by DI water until the waste solution was colorless. Finally, 95% (v/v) ethanol was used to elute biofilm-linked dyes by shaking at 37 °C for 30 min, which was measured by the absorbance at 570 nm. 25833


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Figure 3. Morphological characterization of the different samples. (a) SEM images and (b) AFM images of pure Ti (a1), Ti-PD (a2, b1, b2), Ti-PDS (a3, a4, b3, b4), Ti-PD-S-Ag (a5, a6, b5, b6), and Ti-PD-S-Ag/g (a7, a8, b7, b8). the cell morphology was determined by CLSM study of the stained actin cytoskeleton. 2.9. Cytocompatibility and Osteogenic Activity Assays. 2.9.1. Cell Culture and Seeding. MC3T3 cells were cultured in αminimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37 °C. At 70% confluence, cells were trypsinizated and collected by centrifugation, resuspended, and inoculated onto the sterile coatings in 24-well plates at a density of 1 × 105 cells/mL.The medium was refreshed every 3 days. 2.9.2. Cell Proliferation and Apoptosis. Cell Counting Kits (CCK8, Dojindo) were used to quantify cell proliferation, as detailed elsewhere,30 based on measurement of mitochondrial activity. Additionally, attachment and spread-out conditions of cells were observed by CLSM images of the stained actin cytoskeleton. For the immunofluorescence study, the samples were rinsed three times with PBS and fixed in 4% PFA/PBS. Fixed cells were permeabilized and counterstained with FITC-phalloidin (1:200, 40 min; Sigma) and DAPI (1:1000, 5 min). The level of cell apoptosis was determined by measuring the extracellular release of lactate dehydrogenase (LDH) at 1, 3, and 5 days with an LDH kit (Abcam, Cambridge, MA), following the manufacturer’ protocol. LDH release at the single-cell level (i.e., cellular LDH activity) was determined by normalizing the total LDH activity to the number of cells. 2.9.3. Intracellular ROS Levels. As described in section 2.8.5 for intracellular ROS of bacteria test, the levels of intracellular ROS for MC3T3 cells were also analyzed via a DCFH-DA-based fluorescence method. 2.9.4. Cellular Live/Dead Staining. After culture for 24 h, the survival state of cells on SF-based coatings was observed by staining living cells with 2 μM Calcein AM and the dead cells with 4 μM PI (Live/Dead Cell Stains, Dojindo, Kumamoto, Japan), after which confocal fluorescence images were recorded. 2.9.5. Alkaline Phosphatase (ALP) Activity. For alkaline phosphatase (ALP) activity, after 3, 7, and 14 days, cells on specimens

were rinsed with PBS 3 times and lysed with 1% Triton X-100 for 1 h, and then the cell lysis was mixed with substrate p-nitrophenylphosphate (pNPP; Jiancheng Biotech, Nanjing, China), transforming into p-nitrophenol (pNP) by a ALP enzymatic reaction. Absorbance at 520 nm was read after colorimetric reaction. The ALP activity was normalized against total cellular proteins, as determined by BCA reaction (see section 2.6), and expressed in U per gram of protein. Qualitatively, after fixation of cells with 4% PFA, the specimens were stained with a BCIP/NBT ALP Color Development Kit (Beyotime, China). 2.9.6. Extracellular Matrix (ECM) Collagen and Calcium Assays. Histochemical dyes Sirius Red (SR, 0.1%; Sigma) and Alizarin Red S (ARS, 2%; Sigma), which combine with ECM collagen and calcium salts, were occupied to follow 14 and 28 days cultures, respectively. For staining, cells were fixed in 4% PFA and rinsed with PBS, and 500 μL of SR or ARS dye solution was added and incubated for 18 h or 15 min, respectively; after thorough washing, specimens were dried and photographed. Quantitatively, dyes were extracted using 50% 0.2 M NaOH/methanol (for SR) and 10% cetylpyridinium chloride (for ARS), and the samples were then read on a microplate at 570 or 562 nm. 2.10. Statistical Analysis. All data in this study were analyzed using SPSS 19.0 software. Statistical significance was determined using one-way analysis of variance (ANOVA) or Student’s t test and defined as a p value of less than 0.05. Data values were expressed as mean ± standard deviation.

3. RESULTS 3.1. Formation of SF-AgNPs-Gen Composite Solution. It has been reported that silver nanocolloids were prepared successfully by SF in situ reduction at room temperature with UV irradiation,21 in which the Tyr residues acted as the reduction agent, and the schematic mechanism was shown in the red frame of Figure 1a. As illustrated in Figure S1, the initial 25834


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Figure 4. Analysis of chemical composition for the SF-based coatings. (a) Micro-FTIR spectra; (b) magnified spectra of the quadrate area in image a; (c) survey spectra of XPS; (d and e) XRD patterns.

SF solution was colorless, while after addition of AgNO3 and Gen powders and exposure to UV for 1 h, the color gradually turned to brown. Dependent on the size and shape of particles, color changes of the solution were ascribed to the plasmon resonance (SPR) of AgNPs in the visible region. It was further verified by UV−vis spectra, in which the SPR band at 450 nm represented the formation of AgNPs (Figure. 2a). The most obvious band at 450 nm appeared in Figure 2a4 after 5 h UV irradiation and [SF] = 1 wt %, which meant dilute SF solution and long UV exposure benefited for AgNPs production. Apparently, the addition of Gen had influences on SPR of AgNPs, which may be attributed to the formation of hydrogen bonding between AgNPs and Gen. Also, Figure 2a discloses the influence of the Ag+/SF molar ratio on the amounts and sizes of AgNPs. Compared with 5 wt % SF solution, the absorption bands of AgNPs were red shifted and had stronger intensities in 2 wt % SF solution for the same UV irradiation periods due to the increase of the Ag+/SF molar ratio, implying that the contents and sizes of the nanoparticles in the solutions increase. The band around 300 nm corresponded to SF, which was helpful to elucidate the conformational changes during the SF regeneration and AgNPs formation. Overlap of the red line and black line indicated UV irradiation had no influence on SF solution. Compared to the original SF solution, SF/AgNPs composites showed an obvious signal shift, while SF/AgNPs/Gen displayed an apparent difference compared with SF/AgNPs, which could be ascribed to the electrostatic interactions between Gen and SF. The amounts and particle sizes of the AgNPs were observed from TEM images (Figure 2b). In both composite solutions, extended irradiation time could obviously get smaller sized particles, 10.6 ± 1.8 nm (obtained by analyzing 50 nanoparticles). Interestingly, compared to SF/AgNPs (Figure 2b5), more AgNPs were produced in SF/AgNPs/Gen composites (Figure 2b7), which clearly revealed that the addition of Gen improved the reductive efficiency of SF. The dark particles, appearing in TEM images, with high electron cloudy density and diffraction rings were assigned to AgNPs, while the

surrounding gray substance should be SF. Thus, this suggested that SF could act not only as a reducing agent to reduce Ag+ to AgNPs but also as a stabilizer to ensure uniform distribution of AgNPs by complicated interactions between them.31 3.2. Fabrication and Characterization of the SF-Based Coatings. In the process presented in Figure 1b, Ti−O bonding-enriched pure Ti surface was placed into a pH 8.5 dopamine solution to spontaneously implement PD layer deposition. The resulting surfaces were subsequently impregnated into different composite solutions to form SF-based film by a facile dipping process. Then specimens were put into a drying oven for 15 min at 60 °C, transforming the amorphous structure of SF into a β-sheet crystalline structure to keep the SF-based film stable. To acquire our desired coatings, a circular dipping−drying procedure was cycled 6 times; then its microstructure and physical−chemical properties were characterized. SEM and three-dimensional (3D) AFM, first, were applied on the bare Ti and the one with SF-based films, respectively, for the analysis of their surface properties (Figure 3). The pure titanium surface had a microrough topography with obvious parallel scratches evidently caused by polishing treatments (Figure 3a1), whereas a relatively flat surface was observed for PD film (Figure 3a2), in which the surface roughness was 7.5 ± 0.5 nm (Figure 3b2). Randomly distributed fibrils (yellow arrows) about 10 μm in size existed on the surface of SF-based films (Figure 3a3, 3a5, and 3a7), and white particles with a wider size distribution (100−150 nm) were observed from the high-resolution images which might be micelles self-assembled by SF molecules, the size of which was larger than AgNPs (Figure 3a4, 3a6, and 3a8). The formation mechanisms of micelles and fibrils have been verified as follows:32 SF molecules first aggregated and formed micelles where the surface consists of hydrophilic blocks while the interior is filled with hydrophobic blocks. The formed micelles were then able to aggregate to form fibrils owing to the increasing concentrated solution and the electrostatic forces. Specifically, the negatively charged micelles were stable and well dispersed 25835


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further verified by X-ray diffraction (Figure 4d and 4e). Although the broad distinctive peaks (35−43°) were observed for samples indicative of TiO2 in the form of anatase (A) and rutile (R), there was a detective peak around 21°, corresponding to the crystal domain in β-sheets structure. Compared with the sample of pure SF with 30% β-sheets content, there was no characteristic peak for PD-S and peaks of PD-S-Ag and PD-S-Ag/g were relatively weak, attesting to the improvement on the formation of the silk II crystal structures in the SF-based coatings achieved by the addition of AgNPs and Gen. Moreover, in the diffraction diagram of Ti-PD-S-Ag and Ti-PD-S-Ag/g samples, the Ag-associated characteristic peaks (at 38°, 44.4°, and 77.5°) were hard to be separated from those strong peaks assigned to Ti, due to the low content. Associating these with the above-mentioned results, it can be concluded that a novel AgNPs/Gen-loaded silk fibroin coatings has been successfully developed. 3.3. Protein Adsorption. The protein adsorption ability of the biomaterial surface influences the interaction with cells and to some extent governs the cell attachment due to the combination of integrin and preadsorbed serum proteins.34 As shown in Figure 5a, all SF-based coatings favorably promoted the adsorption of BSA compared to bare Ti and PD film. Protein adsorption is strongly influenced by three main factors: surface chemistry, wettability, and topography (roughness). Here, silk fibroin, as a versatile protein biomaterial, is more attractive to protein than bare Ti, thus favoring the retention of the protein. Compared to Ti-PD-S (Ra = 5.3 ± 0.02 nm), AgNPs-contained coatings had a rougher surface and perhaps a greater capacity for holding protein because it could also adsorb proteins through hydrophobic interactions, electrostatic forces, and hydrogen bonding. It should be noted that the adsorption capability of Ti-PD-S-Ag was far lower than Ti-PD-S and TiPD-S-Ag/g. 3.4. Wettability. In a biological system, the hydrophilicity/ hydrophobicity of the biomaterial surfaces strongly influence the protein adsorption, cell adhesion, dispersion, and proliferation. The wettability of bare Ti- and SF-based coatings was measured by the contact angle (CA) method, as displayed in Figure 5b. High CA values represent hydrophobicity, and low angles indicate hydrophilicity.35 On the basis of the CA results, after formation of PD thin film, the hydrophilicity was impressively improved as contact angle decrease from 54.2° to 35.6°. As mentioned before, the β-sheets structure of SF served as an insoluble part, so the SF-based coatings were hydrophobic and the average CA was 75.2°. Another possible reason causing high hydrophobicity was the formation of fibrils, increasing the roughness of the surface.36 Compared to Ti-PD-S and Ti-PD-SAg, Ti-PD-S-Ag/g presented a little higher CA value. 3.5. Ag+ and Gen Release in PBS. Ag+ detached from AgNPs had great impacts on antibacterial and biocompatible abilities of SF-based coatings, so it was essential to investigate the release profiles of Ag+. Figure 6a and 6b displays the Ag+ release profiles from SF-based coatings in PBS. The total Ag+ content of PD-S-Ag/g coating was 8.39 ± 0.16 μg/cm2. As a control, the Ag+ content of PD-S/Ag was only 7.01 ± 0.21 μg/ cm2, further verifying that more AgNPs were reduced with the addition of Gen, which apparently improved the reduction efficiency of SF. For PD-S-Ag and PD-S-Ag/g coatings, ∼2.98 (a fraction of 41.9%) and ∼4.33 μg/cm2 (a fraction of 51.6%) of Ag+ were released into PBS in the initial 6 h with an average rate of 0.426 and 0.618 μg/cm2 /h, respectively. The accumulated release amounts then were about 3.55 (49.9% of

in dilute solution due to the electrostatic repulsion forces among them, while in concentrated solution, the micelles were forced to alter their configuration to overcome the repulsion forces, resulting in aggregating to form fibrils. In addition, a stable SF structure (silk II) was reported inside SF thin films,33 which is caused by the drying step where SF was stabilized, leading to elasto-plastic behaviors of the films. All this could be the explanation for the tendency of SF to form micelles and further to form fibrils during the process of coating formation. It should be noted that relatively bulky fibrils were observed on the surface of Ti-PD-S-Ag/g (Figure 3 a7 and 3a8) compared with that of Ti-PD-S and Ti-PD-S-Ag due to the reason that the positive charges of Gen reduced the repulsive forces of micelles. The surface roughness of Ti-PD-S, Ti-PD-S-Ag, and Ti-PD-SAg/g was about 5.3 ± 0.02, 22.4 ± 0.05, and 18.9 ± 0.04 nm, respectively. According to the Micro-FTIR results (Figure 4a and 4b), similar results were obtained for PD-S, PD-S-Ag, and PD-S-Ag/ g coatings, indicating that the AgNPs and Gen had no significant effects on the functional groups presence and SF conformation. It can be also observed from Figure 4b that the amide signals in 1650 and 1530 cm−1 were related to the silk II conformation, while the signal shown in 1230 cm−1 was associated with the silk I conformation, indicating the coexistence of both conformations. The secondary structure composition of SF-based coatings was determined by Fourier self-deconvolution (FSD). Table 1 provides an overview of the Table 1. Secondary Structure Content of SF-Based Coatings coatings

β-sheets [%] (±2%)

α-helix [%] (±2%)

random coil [[%] (±2%)

S S-Ag S-Ag/g

14.2 18.7 19.6

62.1 57.4 51.9

10.4 8.5 8.2

secondary structures for S, S−Ag, and S−Ag/g coatings. There were almost invisible characteristic peaks assigned to AgNPs and Gen due to their too low amount. However, the addition of AgNPs and Gen increased the β-sheet structure content of SF without functional groups and conformation changes, as shown in Table 1. Figure 4c presents the XPS spectra of SF-based coatings, in which the representative signals corresponding to C 1s, N 1s, and O 1s were observed at 289.3, 399.0, and 531.0 eV, respectively. The intensified nitrogen signal (11.24 atom %, Table 2) was as a result of the amine groups of polydopamine, Table 2. Concentration of the Elemental Composition at the Surfaces of Functionalized Coatings as Determined by XPS substrates

C%

N%

O%

Ag%

Ti%

Ti-PD Ti-PD-S Ti-PD-S-Ag Ti-PD-S-Ag/g

67.55 65.33 61.87 61.95

11.24 15.10 16.89 17.43

20.79 19.57 18.97 18.99

0 0 2.27 1.62

0.42 0 0 0

and as the generation of the PD layer and SF-based coating, the Ti substrate peaks became weaker. Additionally, the chemical states of the Ag were determined by XPS, where signals at ∼368.0 and 374.0 eV in Figure S2 were related to Ag (0) 3d3/2 and Ag (0) 3d5/2, respectively. After the addition of Gen, the content of Ag markedly decreased from 2.27% to 1.62% due to the formation of AgNPs-Gen complexes (Table 2). Besides, the existence of the β-sheets structure in SF-based coatings was 25836


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Figure 5. Surface properties of different samples the assemblies: (a) protein adsorption and (b) hydrophilicity.

Figure 6. Release behavior of silver in PBS. (a) noncumulative and (b) cumulative release curves of Ag+ after immersion at 37 °C for 30 days. (c) EDS images of samples immersed for 1, 3, and 30 days.

total) and 5.01 μg/cm2 (59.6% of total) upon 1 day of immersion and gradually decreased for an extended period. Compared to PD-S-Ag, PD-S-Ag/g coatings released more Ag+ in PBS and the release rate was faster during the initial period time (within 1 day). However, the Ag+ release profiles of both Ti-PD-S-Ag and Ti-PD-S-Ag/g were extremely similar the rest of the time. Further evidence is given by SEM-EDS results in Figures 6c and S4. The content of Ag kept decreasing, and the residual atomic contents were merely 0.16 and 0.27 atom % for Ti-PD-S-Ag and Ti-PD-S-Ag/g after 30 days of immersion in PBS (Figure 6c). The uniform distribution of Ag partly

reflected the stability of SF-based coatings. Even after approximately 1 month of immersion in PBS solution, SFbased coating was still not completely detached from Ti substrate. Similar to the release profiles of Ag+, Gen went through a burst release in the initial 24 h, and then the release gradually decreased and tended to be stable over an extended period of time (Figure S3). The synchronization release of Ag and Gen was beneficial for their synergetic antibacterial effects. 3.6. Antimicrobial Effects of the Coatings. 3.6.1. Synergetic Antibacterial Property of AgNPs and Gen. Recently, a combination of AgNPs and antibiotics has attracted increasing 25837


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with S. aureus, a common pathogenic strain that is associated with over 70% orthopedic infection.38 To investigate the initial antiadhesive ability of SF-based coatings, S. aureus (cocultured with coatings for 4 h, 1 × 108 CFU/mL) were detached from the surface and counted by measuring the mitochondrial activity. Compared to a bare Ti group, the amounts of bacteria adhered on surface dramatically decreased over 95% in both Ti-PD-S-Ag and Ti-PD-S-Ag/g (Figure 8a). Adversely, PD-S obviously increased the adherent bacteria more than 30% compared with bare Ti. In addition, membrane defects and morphologies of bacteria were investigated by SEM observation (Figure. 8d). As observed from the enlarged images, bacteria adhered, proliferated, and aggregated readily on bare Ti and Ti-PD-S with the bacterial membrane remaining intact. However, bacteria immobilizing on AgNPs-loaded coatings, especially in the Ti-PD-S-Ag/g group, presented defective members with wrinkled and dented morphologies. Moreover, the Live/Dead fluorescence images were taken to intuitively present the living state of bacteria. As depicted in Figure 8h, most bacteria cells were alive (green) and tended to aggregate, finally forming a matured biofilm. The amount of individual bacteria in Ti-PD-S-Ag was impressively reduced compared with that in the Ti-PD-S group, but most of the bacteria were active (green) for further multiplication.We cocultured S. aureus (1 × 108 CFU/mL) with SF-based coatings in LB liquid media for 1 day, and the final OD value and turbidity of solution are shown in Figure. 8g. Due to the rapid proliferation of bacteria, the medium liquid became turbid in Ti, Ti-PD-S, and PD-S-Ag groups. Conversely, benefiting from the burst initial release of Ag and Gen, only the bacteria suspension of the PD-S-Ag/g group became transparent, showing excellent synergistic antibacterial activity. To explore the synergistic antibacterial activity of AgNPs and Gen, we conducted a Kirby−Bauer assay, which is a common antibiotic susceptibility test.39 The zone of inhibition (ZOI) of Ti-PD-SAg and Ti-PD-S-Ag/g was around 18 and 29 mm, whereas Ti and Ti-PD-S did not show any bacterial growth inhibition (Figure S5). This result further confirmed that combination of AgNPs and Gen shows better antibacterial efficiency than solely AgNPs. We could forecast that AgNPs/Gen-contained coatings are capably inhibiting the formation of biofilm according to the robustly initial antiadhesive and planktonic-killing activities. To visualize the formation of biofilm on SF-based coating after a sustained 7 day incubation, a crystal violet staining method was taken to investigate antibiofilm ability. As displayed in Figure 8e, the whole surface of bare Ti, PD-S, and PD-S-Ag was covered by intact and dense biofilm, shielding bacteria from bactericides attack. In sharp contrast, most areas of PD-S-Ag/g coating were gray, and bits of dispersed and fragmented biofilms are pointed out by red circles. Quantitatively, PD-SAg/g coating could remarkably inhibit S. aureus biofilm formation over 80% compared with pure Ti, while AgNPsloaded coating presented the same antibiofilm ability with Ti. Inspiringly, obvious antibiofilm dissimilarities between Ti-PDS-Ag and Ti-PD-S-Ag/g confirmed the effective synergistic antibacterial activity of AgNPs and Gen. PD-S-Ag/g coatings had potential to initially suppress bacterial adhesion and kill planktonic bacteria and long term inhibit subsequent biofilm. These, clinically, were able to greatly improve the ability of host immune systems to eradicate the residual bacteria. Also, it could help to treat on time for early-stage infections before evolving into severe infections.

attention to promote the antibacterial efficiency and to overcome the intrinsic drawbacks of bactericides. In this study, a standard plate count method was applied to evaluate the synergetic bactericidal capability of AgNPs and Gen. In short, after 1-day coculture of S. aureus (1 × 108) and SF-based coating, which contained different amounts of AgNPs and Gen, adhesive bacteria were detached from coatings ultrasonically and counted by a standard plat count way. As presented in Figure 7, only if the Gen concentration was over 1000 μg/L,

Figure 7. To test the synergistic antibacterial activities of AgNPs and Gen, SF-based coatings were cultured with S. aureus (1 × 108 CFU/ mL) for 24 h, and then the bacteria are detached to quantitate by a standard spread-plate technique. Note: the concentrations of Gen and Ag+ were the initial concentrations of the solution for coatings; then Gen and reductive AgNPs were embedded in SF-based coatings to test antibacterial activity.

there was almost no visible S. aureus on agar plate. However, it could be observed that the amount of bacterial colony was over 30 even with the Ag+ concentration reaching 120 mM/L, suggesting that the bacteria proliferated rapidly on the surface of AgNPs-contained coatings. It should be also noticed that the antibacterial efficiency of AgNPs was sharply enhanced by combining with Gen 200 μg/L (with no bactericidal effect), and 10 mM/L Ag+ could relentlessly make all bacteria extinct. Therefore, the final concentration of AgNPs and Gen was 10 mM/L and 200 μg/L, far lower than minimum inhibitory concentration (MIC), representing the lowest concentration of bactericides to eradicate bacteria, of AgNPs (over 120 mM/L) and Gen (1000−1200 μg/L), respectively. The significant decreasing of S. aureus on Ti-PD-S-Ag/g compared to purely contained AgNPs or Gen coatings suggested the synergistic bactericidal ability of AgNPs/Gen. 3.6.2. Antiadhesion and Antibiofilm Activities. Biofilms consisting of immobilized microorganism cells clinically establish colonization on implants after orthopedic surgery, resulting in an outburst of infections like osteomyelitis. The development of biofilms is implemented by several steps, starting from the surface attachment of bacteria, cell accumulation, and then conglomeration into microcolonies. After the biofilm maturation, cells will be apart from the biofilms and turn into floating states to trigger the next biofilm formation cycle elsewhere.37 The existence of biofilms isolates and prevents bacteria from antibiotic attacks and host immune responses since the biofilms are established. It has been widely acknowledged that restraining microbial settlement is more sufficient than the treatment on the biofilms after formation.34 In this study, the planktonic-killing and biofilm-inhibition activities of SF-based coatings were investigated by competing 25838


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Figure 8. Antibacterial activities of different samples against S. aureus (1 × 108 CFU/mL). (a) Relative adherence rate of bacteria; (b) biomass of adhered biofilms; (c) ROS quantitative results of the fluorescence densities in f; (d) typical bacterial morphology; (e) biofilm formation (visualized by crystal violet); patches of aggregate biofilms are indicated by circles; (f) DCF fluorescent images for samples representing the ROS levels on different surfaces during bacterial culture; (g) normalized density and turbidity of S. aureus liquid after incubation with SF-based coatings for 24 h at 37 °C; (h) live (green)/dead (red) status; overlaying of red and green can appear green-yellow.

alone, corresponding with the strongest fluorescence observed on the surface of PD-S-Ag/g coatings (Figure 8f). To further investigate the coatings’ ability to protect osteoblasts from the invasion of bacteria, a bacteria−cell cocultured system was built. It can be observed in Figure 9 that MC3T3 cells incubated on PD-S-Ag/g coatings had inferior cytoskeleton organization compared to others for the first 24 h culture, while the situation was completely inversed for 5 day coculture. Cells on Ti-PD-S-Ag/g displayed rich and wellextended cytoskeleton networks, which was not the case for other groups where few cells with almost negligible cytoskeleton were observed. Quantitatively, almost double

One possible bactericidal mechanism is an oxidative pathway due to the excess ROS. The consequences of excess ROS are acknowledged as disruption to the membrane integrity causing devitalization due to oxidation of fatty acids and production of lipid peroxides which can influence respiratory chain reactions. DCFH-DA fluorescence probe was chosen to label intracellular oxidative stress induced by exceeded ROX (Figure 8c and 8f). Evidently, adhesive bacteria on AgNPs-loaded coatings, especially on Ti-PD-S-Ag/g, generated elevated oxidative stress. Ag+, closely related to ROS production, targeted and disrupted several cellular processes.40 As aforementioned in section 3.5, the AgNPs/Gen complexes released more Ag+ than AgNPs 25839


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actin in Ti-PD-S-Ag/g groups compared to others. Except the Ti-PD-S-Ag group, stress fibers continually assembled in the following days, and finally, a cytoskeleton network developed to distribute on the whole surface after 5 days, suggesting that PDS-Ag/g coating did not disrupt the assembly of the cytoskeleton. As shown in Figure S7, the cells on PD-S-Ag/g coating were strongly adhered and well spread with poreassociated filopodia for 1 day culture, while those on PD-S-Ag coating showed sparse adhesion and relatively weak filopodia. Other than that cells on Ti and Ti-PD-S were well extended and homogeneously distributed. To further investigate the cell toxicity of AgNPs-loaded coatings, cellular Live/Dead staining was taken. As indicated in Figure 11a, some cracked bodies of dead MC3T3 cells were stained in red, existing only in AgNPs-contained groups after 1 day incubation. Overall, the worst survival state of cells was observed on the surface of Ti-PD-S-Ag, implying individual Ag nanoparticles were toxic and injurious for attachment and proliferation of cells. Furthermore, due to the reason that the LDH enzyme could be released from defected cell membrane, LDH test was conducted to expose cell survival state. Apparently, cells on Ti-PD-S-Ag showed the highest LDH value, revealing that individual AgNP caused high intracellular stress, irreversibly leading to death of the cell. Conversely, it could be observed that the addition of Gen was efficient to reduce the toxicity of Ag nanoparticles and in favor of attachment and proliferation of cells. According to Figure 11c, PD-S-Ag/g coating favors cell proliferation similar to that of bare Ti, especially for longer culture periods. However, solely AgNPs-containing group showed high toxicity. In light of the oxidative nature of AgNPs, one vital factor mediating cytotoxicity is oxidative stress, which caused mitochondrial injury and cell apoptosis. The intracellular production of ROS was measured (Figure 11d and 11e). Obviously, the PD-S-Ag coating displayed the strongest fluorescence, and so, the highest amount of generated ROS. Therefore, PD-S-Ag coating showed apparent toxicity to MC3T3. While a relatively low ROS value was obtained in the Ti-PD-S-AgNPs/g group, which could contribute greatly to the biocompatibility of Ti-PD-S-AgNPs/g, detailed mechanisms still need to be explored. 3.8. Osteogenic Differentiation Properties. As one early characteristic of osteogenic differentiative potentiality, ALP encourages the formation of bone mineral and displays evident alterations during osteoblastic differentiation. The ALP expression of cells cultured on PD-S-Ag coating was the lowest at days 7 and 14, indicating the toxicity of singular Ag nanoparticles to osteo-differentiation of MC3T3-E1 cells. Inspiringly, the ALP expression of cells on Ti-PD-S-Ag/g showed a slightly higher level than that on bare Ti at day 14, revealing that PD-S-Ag/g coatings were nontoxic to osteogenic differentiation. Moreover, the highest expression on Ti-PD-S surface manifested the beneficial effect of SF, which may be attributed to the collagen-analogous structure of SF owing more hydrophilic groups to adsorb inorganic ions, promoting bone mineral formation. Also, the entire surface of SF-based coatings was stained by BCIP/NBT to visually exploit ALP expression. The degree of the blue color is directly related to the intensity of the ALP signal. Both Ti-PD-S and Ti-PD-S-Ag/ g groups exhibited large staining areas and a deep blue color, showing active ALP-expressing activity, while all staining features were better than those on the Ti-PD-S-Ag. As osteogenic markers at the late stage, collagen secretion and mineral deposition were further assessed by Sirius red (for

Figure 9. Fluorescent images of MCT3T cocultured with S. aureus (1 × 105 CFU/mL) on various surfaces for 1 and 5 days with actin stained with FITC (green) and nuclei stained with DAPI (blue). (Quantitative data in the Supportive information.)

amounts of cells occupied the surface of Ti-PD-S-Ag/g compared to the case for pure Ti, and the least cells were detected on Ti-PD-S-Ag surface (Figure S6). Combined with the previous antibacterial results, it can be concluded that AgNPs/Gen-embedded coatings could protect cells from bacterial invasion and support cell proliferation thanks to its significant antibacterial property. 3.7. Cytocompatibility of SF-Based Coatings. To investigate the influences of SF-based coatings on cell attachment and proliferation, SEM and confocal microscopy were utilized to observe cells (Figure 10). After 6 h cultivation, cell seeded on bare Ti showed well dispersion by outstretching visible lamellipodia, which guided the cell by sensing the substrate. By contrast, cell cultured on PD-S coating had an elongated morphology and smaller area of focal contact, due to its higher hydrophobicity and surface roughness. Interestingly, AgNPs-contained coatings supported fast, tight adhesion and favorable spreading of cells, and some white particles around the cell might be extracellular secretions (Figure 10a). To further reveal interfacial contact between Ag nanoparticles and cells, amplified images of cell-covered and -uncovered area are shown in Figure 10b. Much more nanoscale pores (red circles) through cell bodies were observed distributing evenly on the surface of Ti-PD-S-Ag in comparison with Ti-PD-S-Ag/g, which tended to overlaying AgNPs by a layer of extracellular matrix. Meanwhile, more nanoparticles were detected on the cell-uncovered area of Ti-PD-S-Ag/g, which further verified that Gen improved the reduction efficiency of SF. In other words, cells spread well on SF-based coatings by outstretching the pseudopodia to sense the surface in the early stage and appeared to overlay Ag nanoparticles. Then the capability of MC3T3 cells developing cytoskeleton on SF-based coatings was inspected, as displayed in Figure 10c. After incubating for 3 days, comparatively strong stress fibers were developed by F25840


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Figure 10. Adhesion−spreading morphologies of MCT3T cells cultured on various surfaces for different times. (a) SEM images for 6 h; (b) magnified images of the selected area in figure a; (c) fluorescent images for 3 and 5 days with actin stained with FITC (green) and nuclei stained with DAPI (blue). Note: Green arrows indicate lammellipodia of cells, and yellow arrows indicate silver nanoparticle bound to coatings. Nanoscale pores through cell bodies caused by high ROS are indicated by red circles.

electron-donating property (the detailed reaction route shown in Figure 1a) but also was utilized to disperse and stabilize the produced AgNPs to maintain the stability of the SF-AgNPs composite solution, owing to the established connection between silk fibroin chains and AgNPs by chemical or hydrogen bonding during the AgNPs formation process.41 Fei et al.21 reduced Ag+ by SF to form a SF-AgNPs composite, a stable yellow gelatinous solution, which almost stayed unchanged for more than 1 month both in the dark and in light. This connection between SF and AgNPs guaranteed an even distribution of AgNPs in SF-based coatings, as verified in Figure 6c. Moreover, an arousing phenomenon was observed through TEM and SEM results that the addition of Gen could intensely improve the reduction efficiency of SF. To explore the behind mechanism, we should first understand the interactions among AgNPs, Gen, and SF. (1) SF and Gen: Many kinds of silk fibroin coatings have been designed to deliver antibiotics, owing to its controllable biodegradation, superior mechanical properties, and service in stabilizing drugs.42 Positively charged Gen could be loaded into SF-based coatings by electrostatic and hydrogen bond interactions with SF. (2) SF and AgNPs: As mentioned before, amino acid residues of SF could attach to the surface of AgNPs (soft cations) through chemical bonding. (3) AgNPs and Gen: The specific interactions between AgNP and aminoglycoside antibiotics are still perplexing. However, the formation of antibiotic-AgNPs complexes instead of a simple mixture of individual AgNP and antibiotic has been

collagen) and ARS staining (for calcium), respectively. As displayed in Figure 12d and 12f, PD-S-Ag/g coatings presented the largest staining areas and the deepest red color, indicating vigorous capability of ECM collagen secretion and calcification of osteoblasts. In addition, collagen stains for bare Ti, Ti-PD-S, Ti-PD-S-Ag, and Ti-PD-S-Ag/g were almost the same, corresponding to quantitative data (Figure 12c). However, Ti-PD-S-Ag/g showed the best apt to promote mineral deposition (Figure 12f), corresponding to previous results that low-concentration Ag+ promoted osteogenic differentiation via induction/activation of TGF-β/BMP signaling in cells. As indicated in Figure 12c and 12e, quantitative data supported the foregoing analyses of collagen and calcium stain, implying PDS-Ag/g coatings promoted growth of MC3T3 cells committing to the mature osteoblasts and facilitated calcification.

4. DISCUSSION In this work, a facile bionic approach was introduced to construct collagen-biomimetic SF-based coatings containing AgNPs and Gen, where Ag+ was reduced to AgNPs (∼10 nm) by SF under UV irradiation, with both antibacterial and osteogenic competencies for application in bone-related implants. As shown in Figure 1b, an inflexible and thin PDassisted coating was employed to anchor the SF-based coating on the surface of Ti, which could greatly improve the stability of SF-based coating. SF not only acted as a reducing agent capable to reduce Ag+ to Ag by the Tyr residues with strong 25841


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Figure 11. Cytocompatibility of the silk-based coatings. (a) Live/dead staining for cells; (b) LDH activity; (c) cell viability detected by CCK8; (d) ROS quantitative results of the fluorescence densities in e; (e) DCF fluorescent images for samples represent the ROS levels on different surfaces.

Figure 12. Osteogenic activates of MC3T3 cultured on samples. (a) ALP activity; (b) ALP positive areas of MC3T3 cultured on Ti (b1), Ti-PD-S (b2); Ti-PD-S-Ag (b3); Ti-PD-S-Ag/g (b4) for 7 days; quantification of collagen secretion (c) and calcium deposition (e); coloration of the collagen (COL) secretion (d, purplish red) and calcium (CAL) deposition (f, red or purplish red) on different specimens for 28 days. 25842


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ACS Applied Materials & Interfaces verified by Raman and UV−vis spectroscopy.14 On this basis, possible mechanisms for the improved reduction efficiency of SF by the addition of Gen were hypothesized: (1) As proved, only Tyr of silk fibroin could reduce Ag+ under UV irradiation and Gen, acting as a shelter, could combine with unreductive residues of SF, which greatly improve contacting probabilities of Ag+ and Tyr; (2) Due to the reason that Gen can combine with SF by electrostatic force or hydrogen bond and complex with AgNPs to form Gen/AgNPs composites, Gen could act as a hauler between SF and AgNPs, improving the reduction efficiency of SF. Overall, a facile route was established to prepare SF-AgNPs-Gen composite solutions, which was then transformed into biofunctional coatings with antibacterial and biocompatible properties by a circular dipping−drying processes. As verified by the results, AgNPs/Gen-loaded SF-based coatings possessed impressive capabilities of antiadhesion, planktonic-killing, and biofilm inhibition. Before revealing synergetic effects, we needed to get clues on the specific antibacterial mechanism of AgNPs and Gen. AgNPs were apt to adhere and accumulate on bacterial membrane, leading to structural changes of cell membranes, which rendered bacteria more permeable.43 Besides, Ag+ detached from AgNPs could get through cell membranes and combine with negatively charged functional groups of proteins and DNA, resulting in cell membrane deformation, DNA condensation, and thus cell death.44 AgNPs also contributed to the production of intracellular ROS, which could form a free radical with great bactericidal competency.10 Different from the perplexing bactericidal mechanism of AgNPs, Gen killed bacteria by blocking the transcription process through combining with 16S rRNA.45 On the basis of the aforementioned mechanisms and the demonstrated results, possible synergetic bactericidal mechanisms of AgNPs and Gen were assumed as follows (Figure 13a). (1) Hua et al.14confirmed the formation of AgNPs−antibiotics complexes connected by the O−Ag bond instead of physical mixing of individual AgNP and Gen (Figure 1a, black frame). Compared to individual AgNPs, the complex carried more positive charges to easily encounter with negatively charged cell membranes, causing a stronger and closer bonding by electrostatic forces, as shown in Figure 13a1. (2) As confirmed by the release curves, Ti-PD-S-Ag/g liberated more Ag+ in the initial state (6 h) than Ti-PD-S-Ag, causing a locally high Ag+ concentration. Therefore, the bacteriumattached complexes released more Ag+ than solely AgNPs under the same condition.14 To some extent, the similar release profiles of Gen and Ag+ were beneficial to their synergetic effects, meanwhile creating provisional and locally high Ag and Gen concentrations near the bacterial surface (Figure 13a2). (3) AgNPs increased the cell membrane permeability, causing a high intracellular concentration of Gen, which plays a vital role in bactericidal capability (Figure 13a3). (4) AgNPs could promote the bactericidal efficiency of Gen by inhibiting assembly of resistance-related enzymes or straight stopping the enzymatic processes involved in antibiotic hydrolysis. (5) As shown in Figure 8f, Ti-PD-S-Ag/g induced more intracellular ROS compared with other groups, which acted as a pivotal reason for synergetic antibacterial activities. Jose Ruben Morones-Ramirez et al. found that intracellular ROS caused by Ag treatment could be harnessed to enhance the killing efficacy of antibiotics in vitro and in vivo.46 Meanwhile, Wang et al. proved that ROS produced in solution contributed to kill the bacteria based on electron transfer theory.47 Consequently, the

Figure 13. Schematic illustration of the possible antibacterial (a) and biocompatible (b) mechanism on the AgNPs/Gen-contained SF-based coatings.

Gen-AgNPs complexes were probably responsible for extracellular ROS production in which AgNP improved the electron transfer. The production of extracellular ROS is schematically illustrated in Figure 13a. It has been reported that the fast release and storage of toxic Ag+ were the two main reasons for the mammal cell toxicity of AgNPs.17,48 Ag+ was able to penetrate through the cell membrane and then react with protein and DNA, leading to their dysfunction.18 Recent studies also revealed that the aggregation of AgNPs on the cell membrane generated a large amount of reactive oxygen species (ROS), resulting in cell apoptosis. Additionally, the sharp edges of AgNP were apt to damage the cell membrane when contacting with cells directly.49 As verified before, AgNPs/Gen-embedded coatings were approved for the attachment and proliferation of cells, exhibiting no apparent cell toxicity. As illustrated in Figure 13b, the concentration of AgNPs loaded into coatings was quite low (6 mM/L) due to its synergistic effects with Gen. In vitro, the cytotoxicity effects of AgNPs on MC3T3 cells was concentration sensitive and could only promote attachment and proliferation at a very low concentration.48 This result concurred with the conclusions of Zhang et al. that AgNPs induced MSCs activation (proliferation, cytokines release, and chemotaxis) at nontoxic concentration.48 Even though the specific mechanism has not been confirmed, some studies revealed that AgNPs could enhance cell viability by interacting 25843


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coatings simultaneously promoted the antibacterial activity, cellular compatibility, and osteogenic properties of orthopedic titanium with broad application prospects.

with DNA and activating expression of genes such as HIF-1α and IL-8, which in turn enhanced cell proliferation.50 Through TEM images, dispersive AgNPs were fully orbicular with a diameter of around 10 nm, which means the cell death caused by sharp edges was negligible. Furthermore, packaged AgNPs by SF chains availably inhibited their toxicity, since SF could slow down the release of Ag+ as well as prevent the direct interaction between AgNPs and cell membrane. Overall, the good biocompatibility of the PD-S-Ag/g coatings should owe to the combination of low-concentration Ag and biomimetic SFbased coatings. The cells proliferation could continuously promote collagen secretion and ALP expression, indicating cultured medium became alkaline to further facilitate bone formation, especially the formation of a mineralized matrix after approximately 1 month culture. It could be concluded from ALP, collagen, and calcium results that PD-S-Ag/g coatings were beneficial to osteogenic activity due to the following reasons: (1) the amorphous and crystal substructures in the β-sheet strikingly resemble Col I, which acted as nucleation sites for the deposition of inorganic nanocrystals;25,26 (2) SF film supported firm adhesion of human osteoblast cells with extended filopodia and lamellopodia, which were considered as a vital step in osteanagenesis; (3) In aqueous condition, precipitated SF molecular chains stretched into a β-sheet, exposing more hydrophilic groups on the outside of coatings to adsorb mineral ions to form mineralized nodules;23,24 (4) At the osteogenic genetic level, SF efficiently suppressed Notch-activated genes and subsequently blocked the activation of Notch-specific reporter, resulting in upregulation of osteogenic markers.51 Furthermore, AgNPs were identified to encourage osteogenic activities of MSCs and to promote bone fracture healing in vivo.48 When the concentration of AgNPs was lower than 10 μM, it promoted mMSCs’ differentiation toward osteoblast by inducing and activating TGF-β/BMP signaling, which further induced osteogenesis of MSCs. Even if the AgNPs/Gen-loaded SF-based coatings exhibited excellent compatibility and osteogenesis in the short-term evaluation (less than 1 month), investigation of the potential risk of long-term in vivo evaluation still needs to be done. Overall, AgNPs combining with Gen to show excellently synergistic activity were loaded in biomimetic-designed SF coating with inspiring antibacterial ability, biocompatibility, and osteogenic activities.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06757. Color change of different composite solutions, corresponding core-level spectra for Ag 3d and C 1 s, release behavior of Gen in PBS, surface observation of samples after immersion, viability of cell cocultured with bacteria, inhibitory zones for the samples against S. aureus, interfacial interplay between MC3T3 cells and SFbased coatings (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 0086-10-6275 3404. E-mail: [email protected]. ORCID

Ming Li: 0000-0001-5823-8108 Yan Cheng: 0000-0001-7956-9395 Yufeng Zheng: 0000-0002-7402-9979 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was jointly supported by the National Natural Science Foundation of China (Nos. 31670974 and 31370954), the Project of Scientific and Technical Plan of Beijing (No. Z141100002814008), and Beijing Natural Science Foundation (2164073).

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REFERENCES

(1) Long, M.; Rack, H. J. Titanium Alloys in Total Joint ReplacementA Materials Science Perspective. Biomaterials 1998, 19, 1621−1639. (2) Challa, V. S. A.; Mali, S.; Misra, R. D. K. Reduced Toxicity and Superior Cellular Response of Preosteoblasts to Ti-6Al-7Nb Alloy and Comparison with Ti-6Al-4V. J. Biomed. Mater. Res., Part A 2013, 101, 2083−2089. (3) Zhang, Y.; Hou, C.; Yu, S.; Xiao, J.; Zhang, Z.; Zhai, Q.; Chen, J.; Li, Z.; Zhang, X.; Matti, L. IRAK-M in Macrophages Around Septically and Aseptically Loosened Hip Implants. J. Biomed. Mater. Res., Part A 2012, 100, 261−268. (4) Braem, A.; Van Mellaert, L.; Mattheys, T.; Hofmans, D.; De Waelheyns, E.; Geris, L.; Anné, J.; Schrooten, J.; Vleugels, J. Staphylococcal Biofilm Growth on Smooth and Porous Titanium Coatings for Biomedical Applications. J. Biomed. Mater. Res., Part A 2014, 102, 215−224. (5) Ma, M.; Kazemzadeh-Narbat, M.; Hui, Y.; Lu, S.; Ding, C.; Chen, D. D. Y.; Hancock, R. E. W.; Wang, R. Local Delivery of Antimicrobial Peptides Using Self-organized TiO2 Nanotube Arrays for Peri-Implant Infections. J. Biomed. Mater. Res., Part A 2012, 100A, 278−285. (6) Stigter, M.; Bezemer, J.; de Groot, K.; Layrolle, P. Incorporation of Different Antibiotics into Carbonated Hydroxyapatite Coatings on Titanium Implants, Release and Antibiotic Efficacy. J. Controlled Release 2004, 99, 127−137. (7) Mei, S.; Wang, H.; Wang, W.; Tong, L.; Pan, H.; Ruan, C.; Ma, Q.; Liu, M.; Yang, H.; Liang, Z. Antibacterial Effects and Biocompatibility of Titanium Surfaces with Graded Silver Incorporation in Titania Nanotubes. Biomaterials 2014, 35, 4255−4265. (8) Shi, Z.; Neoh, K. G.; Kang, E. T.; Poh, C.; Wang, W. Bacterial Adhesion and Osteoblast Function on Titanium with Surface-grafted

5. CONCLUSIONS In this study, AgNPs/Gen-loaded SF-based coatings were successfully fabricated on Ti implants by a circular dipping− drying procedure. AgNPs with a diameter of around 10 nm and spherical morphologies were produced by reducing Ag+ with SF, which also stabilized AgNPs to ensure a uniform distribution. Due to the efficient synergetic activity of AgNPs and Gen, the PD-S-Ag/g coatings significantly inhibited bacteria growth, adhesion, and formation of biofilm. Moreover, it evidently improved the attachment and proliferation of MC3T3 cells and simultaneously inhibited bacteria proliferation in a cell−bacteria coculture system. The AgNPs/Gencontained coatings supported the growth of MC3T3 cells and benefitted osteoblast differentiation compared with purely AgNPs-embedded coatings. There was no statistically significant difference in LDH and ROS activity between pristine Ti and AgNPs/Gen-loaded coatings, which indicated that these biofunctional coatings were nontoxic compared to Ti. In summary, these AgNPs/Gen-loaded biomimetic SF-based 25844


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