Gentamicin-Embedded Silk

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06757 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017

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ACS Applied Materials & Interfaces

Bioinspired and Biomimetic AgNPs/gentamicin-embedded Silk Fibroin Coatings for Robust Antibacterial and Osteogenetic Applications Wenhao Zhoua, Zhaojun Jiaa, Pan Xionga, Jianglong Yana, Yangyang Lia, Ming Lic, Yan Chenga,*, Yufeng Zhenga,b

a

Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China

b

Department of Advanced Materials and Nanotechnology, College of Engineering, Peking University, Beijing

100871, China

c

China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China

*Corresponding author Y. Cheng, Ph. D. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China Tel&Fax: 0086-10-6275 3404 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract

With the progressively increasing demand for orthopedic Ti implants, the balance realization between two primary complications restricting implants applications is imperative needed to be solved that the lack of bone tissue integration and biomedical device-associated infections (BAI), where emergence of multi-resistance bacteria make it worse. Notably, a combination of silver nanoparticles (AgNPs) and a kind of antibiotic can synergistically inhibit bacterial growth, where 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+ were 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 d) and tenfold improvement of antibacterial efficiency were achieved for our novel AgNPs and Gen embeded silk fibroin coatings. In bacteria and cells co-cultured system, AgNPs/Gen-embedded 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/Gen-embedded 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 coatings 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


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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 are 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 bactericide-doped coatings,7 bioactive antibacterial polymerization8 and adhesion-resistant coatings.9 Silver-containing coatings has attracted lots of attentions to be applied on BAI inhibition owing to its superior performances 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 12

thiol-containing proteins to impair their activities.

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 multidrug-resistant 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 growing of multidrug-resistant bacteria, while the 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 individually bactericidal behaviors of antibiotics and AgNPs were replaced by the formation of



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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 paper, a biomimetic SF-based coating was established to load antibacterial agents where particulate silver is collaborated with Gen to eradicate bacteria. Silk fibroin extracted from Bombyx mori silkworm is usually applied in biomedical coatings owing to its biocompatible and substrate recognized capabilities. An

environment-friendly and

energy-efficient synthetic route has been reported to form SF-AgNPs composites, involving silk fibroin reducing silver ions, meanwhile, 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 that 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 non-collagenous proteins, acting as nucleation sites for the crystallization of HA-nanocrystals.25-27 Additionally, the secondary structure of silk fibroin β-sheet crystalline 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 non-osteogenic 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 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


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coatings, where β-sheet secondary structure deeply influence physical-chemical properties of SF-based coatings; (3) to assess the synergistic antibacterial activities of AgNPs and Gen contained coatings according to their anti-adhesion, planktonic-killing and anti-biofilm evaluations; (4) to investigate in vitro how osteoblast-like MC3T3 cells react with the AgNPs/Gen-embedded coatings, in term 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.

1. Experimental section

1.1. Preparation of Silk Fibroin (SF) Solution. Sercin, a hydrophilic gelatinous coating protein, helps bonding 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, the sercin removal was accomplished by twice treatment with 0.5 wt % NaHCO3 aqueous solution at 100oC for 30 min, followed by washing with distillation water and drying in the air at room temperature. After degumming, B. mori silk fibers were added into 60 oC 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 oC. The concentration of final SF solution was around 10 wt% and then deionized water was used to dilute to 5 wt%.

1.2.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. Afterwards, an



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UV lamp (40 W, from Philips) was used to expose the SF/AgNO3/Gen solution under the ultra violet 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 (10mm × 10mm × 0.5mm) were mechanically polished up to 2000 grit and rinsed with ultrasonication in acetone, ethanol, and deionized water (DI) in sequence. Then, the discs were dry 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 under constantly vibrating in darkness at 37oC, and then isolate the excess monomer and particles by thoroughly ultrasonication to produce polydopamine-decorated 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 (Fig. 1b). 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.


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The water contact angle on different specimens were determined by the sessile-drop water method, using an 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) were transferred gently onto different specimens and co-incubated 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 follow: samples were placed at the bottom of each well in a 24-well plates. 1 mL 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 oC. The medium collection was conducted after each set time interval, and then the sample was washed with 1mL PBS followed by the addition of 1 mL 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



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incubation at room temperature. Finally, the complex was analyzed as the method mentioned before. The experiment was repeated for three times. 2.8. Antimicrobial Activity Assays. 2.8.1 Bacteria Culture and Inoculation.

Under the 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 co-cultured with specimens (sterilized by ultra violet) 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 color change of LB solution, which could be measured by colorimetrical method. The antibacterial rates for adhered bacteria (Raa) were thus calculated Raa (%) =B/A × 100, where A, 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 (50−100%), the morphologies of anchored bacteria was 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


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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 anti-biofilm ability of SF-based coatings was investigated by incubating with bacteria (1 × 108 CFU/mL) for 10 d. The culture medium was refreshed every 3 d. Firstly, the specimens were rinsed by PBS for 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.

2.8.5 The Formation 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 co-culture of SF-based coatings and bacteria for 1 d, the medium was refreshed by dilute DCFH-DA (non fluorescent) and incubated for another 2 h at 37 °C, forming 2′,7′ dichlorofluorescin (DCF, fluorescent) with 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 Co-culture of S. aureus and MC3T3 Cells. S. aureus was chose as invaded bacteria, which involved in most orthopedic infections .4 At 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 for 3 times. The cells-adhesive coatings were put into new plates containing fresh medium (antibiotic-free α-MEM). Next, 1 mL S. aureus (1 × 105 CFU/mL) were 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 incubated for 1-, 3- and 5-d, cell proliferation was



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quantified using Cell Counting Kits and 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 α-minimum 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 three days.

2.9.2. Cell Proliferation and Apoptosis.

Cell Counting Kits (CCK-8, Dojindo) was used to quantify cell proliferation, as detailed elsewhere,30 based on the 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 d 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.


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2.9.4. Cellular Live/Dead Staining.

After culture for 24 h, the survival state of cells on SF-based coatings were 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-d, cells on specimens were rinsed with PBS for 3 times and lysed with 1% Triton X-100 for 1 h, and then the cell lysis was mixed with substrate p-nitro phenylphosphate (pNPP; Jiancheng Biotech, Nanjing, China), transforming into p-nitro-phenol (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 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 a 14 d and 28 d 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 ap value of less than 0.05. Data values were expressed as mean ± standard deviation.



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3. Results

3.1 Formation of SF-AgNPs-Gen Composite Solution It has 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 red frame of Fig. 1a. As illustrated in Fig. S1, the initial 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 size and shape of particles, color changes of the solution were ascribed to 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 Fig. 2 a4, 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, Fig. 2a disclosed the influence of Ag+/SF molar ratio on the amounts and sizes of AgNPs. Compared with 5 wt% SF solution, the absorption bands of AgNPs got red shift and stronger intensities in 2 wt% SF solution for the same UV irradiation periods due to the increase of 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 red line and black line indicated UV irradiation had no influence on SF solution. Compared to original SF solution, SF/AgNPs composites showed obvious signal shift, while SF/AgNPs/Gen displayed apparent difference compared with SF/AgNPs, which could ascribe to the electrostatic interactions between Gen and SF. The amounts and particle sizes of the AgNPs were observed from TEM images (Fig. 2b). In both composite solutions, extended irradiation time could obviously get smaller size particles, 10.6±1.8 nm (obtained by analyzing 50 nanoparticles). Interestingly, compared to SF/AgNPs (Fig. 2 b5), more AgNPs were produced in SF/AgNPs/Gen composites (Fig. 2b7), which clearly revealed that the addition of Gen


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improved the reductive efficiency of SF. The dark particles, appeared 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

Fig.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. The scheme is not in real scale.



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Fig.2. (a) UV-vis spectra of the SF/AgNPs and SF/AgNPs/Gen composite solution with different UV irradiation time (1 h or 5 h) and SF solution concentration (2 wt% and 5 wt%). (b) TEM images of the SF/AgNPs and SF/AgNPs/Gen composites with different UV irradiation time (1 h or 5 h).

3.2 Fabrication and Characterization of the SF-based Coatings In the process presented in Fig. 1b, Ti-O bonding enriched pure Ti surfaces was placed into a pH 8.5 dopamine solution to spontaneously implement PD layer deposition. The resulting


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surfaces were subsequently impregnated into different composite solutions to form SF-based film by a facile dipping process. Then, specimens were put into drying oven for 15 min at 60oC, transforming amorphous structure of SF into β-sheet crystalline structure to keep SF-based film stable. To acquire our desired coatings, a circular dipping-drying procedure was cycled for 6 times, then its microstructure and physical-chemical properties were characterized. SEM and three-dimensional (3D) AFM, firstly, were applied on the bare Ti and the one with SF-based films, respectively, for the analyzation of their surface properties (Fig. 3). The pure titanium surface had a micro-rough topography with obvious parallel scratches evidently caused by polishing treatments (Fig. 3a1), whereas a relatively flat surface was observed for PD film (Fig. 3 a2), in which the surface roughness was 7.5±0.5 nm (Fig. 3 b2). Randomly distributed fibrils (yellow arrows) about 10 µm in size existed on the surface of SF-based films (Fig. 3 a3, a5, a7) 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 (Fig. 3 a4, a6, a8). The formation mechanisms of micelles and fibrils have been verified as follow32: SF molecules firstly aggregated and formed micelles where surface consists of hydrophilic blocks while interior 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 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 films33, 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 process of coating formation. It should be noted that relatively bulky fibrils were observed on the surface of Ti-PD-S-Ag/g (Fig. 3 a7, a8) 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-S-Ag/g was about 5.3±0.02, 22.4±0.05 and 18.9±0.04 nm, respectively.



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Fig.3. Morphological characterization of the different samples. (a) SEM images and (b) AFM images of pure Ti (a1), Ti-PD (a2, b1, b2), Ti-PD-S (a3, a4, b3, b4), Ti-PD-S-Ag (a5, a6, b5, b6), Ti-PD-S-Ag/g (a7, a8, b7, b8).

According to the Micro-FTIR results (Fig 4a, b), 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 Fig. 4b that the amide signals in 1650 cm−1 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 provided an overview of the secondary structures for S, S-Ag and S-Ag/g coatings, respectively. There were almost invisible characteristic peaks assigned to AgNPs and Gen due to their too low amount. But 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. Fig. 4c presented the XPS spectra of SF-based coatings, in which the representative signals corresponding to C1s, N1s


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and O1s was observed at 289.3 eV, 399.0 eV and 531.0 eV respectively. The intensified nitrogen signal (11.24 at. %, Table 2) was as result of amine groups of polydopamine, and as the generation of 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 Fig. 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 β-sheets structure in SF-based coatings was further verified by X-ray diffraction (Fig. 4d and e). Although the broad distinctive peaks (35o - 43o) 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 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.



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

Table.1. Secondary structure content of SF-based coatings

Coatings

Beta-sheets [%] (±2%)

Alpha-Helix [%] (±2%)

Random coil [%] (±2%)

S

14.2

62.1

10.4

S-Ag

18.7

57.4

8.5

S-Ag/g

19.6

51.9

8.2


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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

67.55

11.24

20.79

0

0.42

Ti-PD-S

65.33

15.10

19.57

0

0

Ti-PD-S-Ag

61.87

16.89

18.97

2.27

0

Ti-PD-S-Ag/g

61.95

17.43

18.99

1.62

0

3.3 Protein Adsorption The protein adsorption ability of biomaterial surface influences the interaction with cells, and in some extent governs the cell attachment, due to the combination of integrin and preadsorbed serum proteins.34 As shown in Fig. 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 adsorption capability of Ti-PD-S-Ag was far lower than Ti-PD-S and Ti-PD-S-Ag/g.



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3.4 Wettability In biological system, 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 contact angle (CA) method, as displayed in Fig. 5b. High CA values represent hydrophobicity, and low angles indicate hydrophilicity.35 Based on the CA results, after formation of PD thin film, the hydrophilicity was impressively improved as contact angle decrease from 54.2o to 35.6o. As mentioned before, the β-sheets structure structure of SF served as an insoluble part, so the SF-based coatings were hydrophobic and the average CA was 75.2o. 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-S-Ag, Ti-PD-S-Ag/g presented a little higher CA value.

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


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Fig.6. The release behavior of silver in PBS. (a) non-cumulative and (b) cumulative release curves of Ag+ after immersion at 37oC for 30 d. (c) show EDS images of samples immersed for 1, 3 and 30 d respectively.

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+. Fig. 6a, 6b displayed 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, 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 reduction efficiency of SF. For PD-S-Ag and PD-S-Ag/g coatings,



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~2.98 µg/cm2 (a fraction of 41.9%) and ~4.33 µg/cm2 (a fraction of 51.6%) of Ag+ was released into PBS in the initial 6 h with an average rate of 0.426 µg/cm2/h and 0.618 µg/cm2/h, respectively. The accumulated release amounts then were about 3.55 µg/cm2 (49.9% of total) and 5.01 µg/cm2 (59.6% of total) upon 1 d immersion, and gradually decreased for an extended periods. Compared to the PD-S-Ag, PD-S-Ag/g coatings released more Ag+ in PBS and release rate was faster during the initial period time (within 1 d). However, the Ag+ release profiles of both Ti-PD-S-Ag and Ti-PD-S-Ag/g were extremely similar in the rest of time. Further evidence was given by SEM-EDS results in Fig. 6c and S4. The content of Ag kept decreasing and the residual atomic contents were merely 0.16 at. % and 0.27 at. % for Ti-PD-S-Ag and Ti-PD-S-Ag/g after 30 d immersion in PBS (Fig. 6c). The uniform distribution of Ag partly reflected the stability of SF-based coatings. Even after approximate one-month immersion in PBS solution, SF-based 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 (Fig. S3). The synchronization releases 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 attracted increasing attentions 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 one-day co-culture of S. aureus (1×108) and SF-based coating, which contained different amounts of AgNPs and Gen, adhesive bacteria were detached from coatings by ultrasonic and counted by a standard plat count way. As presented in Fig. 7, only if the Gen concentration was over 1000 µg/L, 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 the Ag+ concentration reaching 120 mM/L, suggesting that the bacteria proliferated rapidly on the surface of AgNPs-contained coatings. It should be also


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noticed that the antibacterial efficiency of AgNPs sharply enhanced by combining with Gen 200 µg/L (with no bactericidal effect), and 10 mM/L Ag+ could relentlessly extinct all bacteria. 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 significantly 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.

Fig.7. To test 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.



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3.6.2 Anti-adhesion and Anti-biofilm Activities

Biofilms consisted of immobilized microorganism cells clinically establish colonization on implants after orthopedic surgery, resulting in outburst of infections like osteomyelitis. The development of biofilms implements by several steps, starting from the surface attachment of bacteria, cell accumulation, and then conglomeration into micro colonies. After the biofilm maturation, cells will be apart from the biofilms and turn into floating states to trigger next biofilm formation cycle elsewhere.37 The existence of biofilms isolates and prevents bacteria from antibiotic attacks and host immune responses since the biofilms established. It has been widely acknowledged that restraining microbial settlement is much 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 with S. aureus, a common pathogenic strain that is associated with over 70% orthopedic infection.38 To investigate the initial anti-adhesive ability of SF-based coatings, S. aureus (co-cultured with coatings for 4 h, 1 × 108 CFU/mL) were detached from surface and counted by measuring mitochondrial activity. Compared to 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 (Fig. 8a). Adversely, PD-S obviously increased the adherent bacteria more than 30% compared with bare Ti. In addition, membrane defects and morphologies of bacteria was 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 bacterial membrane remaining intact. However, bacteria immobilizing on AgNPs-loaded coatings, especially in 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 Fig. 8h, most bacteria cells were alive (green) and attended to aggregate, finally forming a matured biofilm. The amounts of individual bacteria in Ti-PD-S-Ag was impressively reduced compared with that in Ti-PD-S group, but most of bacteria were active (green) for further multiplication.We co-cultured S. aureus (1×108 CFU/mL) with SF-based coatings in LB liquid media for 1 d, and the final OD value and turbidity of solution were shown in Figure. 8g. Due to the rapid proliferation of bacteria, the medium liquid turned to be


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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 PD-S-Ag/g group turned to be 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-S-Ag and Ti-PD-S-Ag/g were around 18 mm and 29 mm, whereas Ti and Ti-PD-S didn’t show

any bacterial growth inhibition (Fig. S5). This results further confirmed that combination of AgNPs and Gen show 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 anti-adhesive and planktonic-killing activities. To visualize the formation of biofilm on SF-based coating after a sustained 7 d incubation, a crystal violet staining method was taken to investigate anti-biofilm ability. As displayed in Fig. 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 exhibited grey, and a bits of dispersed and fragmented biofilms were pointed out by red circles. Quantitatively, PD-S-Ag/g coating could remarkably inhibit S. aureus biofilm formation over 80% compared with pure Ti, while AgNPs-loaded coating presented the same anti-biofilm ability with Ti. Inspiringly, obvious anti-biofilm dissimilarities between Ti-PD-S-Ag and Ti-PD-S-Ag/g confirmed effective synergistic antibacterial activity of AgNPs and Gen. PD-S-Ag/g coatings had a potential to initially suppress bacterial adhesion and kill planktonic bacteria, and long-termly 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 the early-stage infections before evolving into severe infections. One possible bactericidal mechanism is an oxidative pathway due to the excess ROS. The consequences of excess ROS is acknowledged as disruption to the membrane integrity causing devitalization due to the oxidation of fatty acids and the 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 (Fig. 8c, f). Evidently, adhesive



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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 alone, corresponding with the strongest fluorescence observed on the surface of PD-S-Ag/g coatings (Fig. 8f). To further investigate coatings’ ability to protect osteoblasts from the invasion of bacteria, a bacteria-cell co-cultured system was built. It can be observed in Fig. 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 d co-culture. Cells on Ti-PD-S-Ag/g displayed rich and well-extended cytoskeleton networks, which was not the case for other groups where few cells with almost negligible cytoskeleton was observed. Quantitatively, almost double 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 (Fig. 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 significantly antibacterial property.


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Fig.8. Antibacterial activities of different samples against S. aureus (1×108 CFU/mL). (a) relative adherence rate of bacteria; (b) the biomass of adhered biofilms; (c) the 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 at37 °C; (h) live (green)/dead (red) status, the overlaying of red and green can appear greenyellow.



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Fig.9. Fluorescent images of MCT3T co-cultured with S. aureus (1×105 CFU/mL) on various surfaces for 1 d and 5 d with actin stained with FITC (green) and nuclei stained with DAPI (blue). (Quantitative data in Supportive information)

3.7 Cytocompatibility of SF-based Coatings To investigate influences of SF-based coatings on cell attachment and proliferation, SEM and confocal microscope were utilized to observe cells (Fig. 10). After 6 h cultivation, cell seeded on bare Ti showed well dispersion by outstretching visible lamellipodia, which guided cell by sensing the substrate. By contrast, cell cultured on PD-S coating had an elongated morphologies and fewer 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 (Fig. 10a). To further reveal interfacial contact between Ag nanoparticles and cells,


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amplified images of cell-covered and -uncovered area were shown in Fig. 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 cell-uncover area of Ti-PD-S-Ag/g, which further verified Gen improved reduction efficiency of SF. In a word, 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 Fig. 10c. After incubating for 3 d, comparatively strong stress fibers were developed by F-actin in Ti-PD-S-Ag/g groups compared to others. Except 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 d, suggesting that PD-S-Ag/g coating did not disrupt the assembly of the cytoskeleton. As shown in Fig. S7, the cells on PD-S-Ag/g coating were strongly-adhered and well-spread with pore-associated filopodia for 1 d culture, while those on PD-S-Ag coating showed sparse adhesion and relatively weak filopodia. Except that, cells on Ti and Ti-PD-S were well-extended and homogeneously-distributed. To further investigate cell toxicity of AgNPs-loaded coatings, cellular Live/Dead staining was taken. As indicated in Fig. 11a, some cracked bodies of dead MC3T3 cells were stained in red, existing only in AgNPs-contained groups after 1 d 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 cell. Conversely, it could be observed that the addition of Gen was efficient to reduce toxicity of Ag nanoparticles and in favor of attachment and proliferation of cells. According to Fig. 11c, PD-S-Ag/g coating favor cell proliferation similar to that of bare Ti, especially for a longer culture periods. However, solely AgNPs contained 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



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measured (Fig. 11d, e). 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, relatively low ROS value was obtained in Ti-PD-S-AgNPs/g group, which could contribute greatly to the biocompatibility of Ti-PD-S-AgNPs/g, but the detailed mechanisms are still needed to be explored.

Fig.10. The adhesion-spreading morphologies of MCT3T cells cultured on various surfaces for different time. (a) SEM images for 6 h; (b) magnified images of the selected area in figure (a); (c) fluorescent images for 3 d and 5 d 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.


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

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 day 7 and 14, indicating the toxicity of singular Ag nanoparticles to osteo-differentiation of MC3T3-E1 cells.



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Inspiringly, the ALP expression of cells on Ti-PD-S-Ag/g showed slightly higher level than that on bare Ti at day 14, revealing that PD-S-Ag/g coatings were non-toxic 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 owning 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 ALP signal. Both Ti-PD-S and Ti-PD-S-Ag/g group exhibited large staining areas and 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, the collagen secretion and mineral deposition were further assessed by Sirius red (for collagen) and ARS staining (for calcium), respectively. As displayed in Fig. 12d and f, 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 same, corresponding with quantitative data (Fig. 12c). However, Ti-PD-S-Ag/g showed the best apt to promote mineral deposition(Fig. 12f), corresponding with previous results that low concentration Ag+ promoted osteogenic differentiation via induction/activation of TGF-β/BMP signaling in cells. As indicated in Fig. 12c, e, quantitative data supported the foregoing analyses of collagen and calcium stain, implying PD-S-Ag/g coatings promoted the growing MC3T3 cells committing to the mature osteoblasts and facilitated calcification.


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Fig.12. The 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 d; the quantification of collagen secretion (c) and calcium deposition (e); The coloration of the collagen (COL) secretion (d, purplish red) and calcium (CAL) deposition (f, red or purplish red) on different specimens for 28 d.

4. Discussion

A facile bionic approach, in this work, was introduced to construct collagen-biomimetic SF-based coatings containing AgNPs and Gen, where Ag+ were 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 Fig. 1b, an inflexible and thin PD assisted 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 electron donating property (the detailed reaction route shown in Fig. 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



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stable yellow gelatinous solution, which almost stayed unchanged for more than one month both in the dark and in light. This connections between SF and AgNPs guaranteed even distribution of AgNPs in SF-based coatings, as verified in Fig. 6c. Moreover, an arousing phenomenon was observed through TEM and SEM results that the addition of Gen could intensely improve reduction efficiency of SF. To explore the behind mechanism, we should firstly understand the interactions among AgNPs, Gen and SF: (1) SF and Gen: A lot kinds of silk fibroin coatings had 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 were still perplexed. However, the formation of antibiotic-AgNPs complexes instead of simple mixture of individual AgNP and antibiotic had been verified by Raman and UV-vis spectroscopy.14 On the basis of these, 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 un-reductive 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 anti-adhesion, 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 to 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 negative charged functional groups of proteins and DNA, resulting in cell membrane deformation, DNA


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condensation and thus cell death.44 AgNPs also contributed to the production of intracellular ROS, which could form free radical with great bactericidal competency.10 Varied from perplexing bactericidal mechanism of AgNPs, Gen killed bacteria by blocking transcription process through combining with 16S ribosomal RNA.45 Based on the aforementioned mechanisms and the demonstrated results, possible synergetic bactericidal mechanisms of AgNPs and Gen were assumed as following (Fig. 13a): (1) Hua et al.14confirmed the formation of AgNPs-antibiotics complexes connected by the O-Ag bonding instead of physical mixing of individual AgNP and Gen (Fig.1a, black frame). Compared to individual AgNPs, the complex carried more positive charges to easily encounter with negative charged cell membranes, causing a stronger and closer bonding by electrostatic forces, as shown in Fig. 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 bacterium-attached 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 a provisional and locally high Ag and Gen concentrations near bacterial surface. (Fig. 13a2); (3) AgNPs increased cell membrane permeability, causing a high intracellular concentration of Gen, which plays a vital role in bactericidal capability (Fig. 13a3). (4) AgNPs could promote 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 Fig. 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 Gen-AgNPs complexes were probably responsible for extracellular ROS production in which AgNP improved the electron transfer. The production of extracellular ROS was schematically illustrated in Fig. 13a. It has been reported that the fast release and the storage of toxic Ag+ were two main reasons for the mammal cell toxicity of AgNPs.17, 48 Ag+ was able to penetrate through cell membrane and then react with protein and DNA, leading to their dysfunction.18 Recent studies also



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revealed that the aggregation of AgNPs on 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 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 Fig. 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 it could only promote attachment and proliferation at 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 the specific mechanism has not been confirmed, some studies revealed that AgNPs could enhance cell viability by interacting 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 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 SF-based coatings.


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



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The cells proliferation could continuously promote collagen secretion and ALP expression, indicating cultured medium turned to be alkaline to further facilitate bone formation, especially the formation of a mineralized matrix after approximately a 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 crystals substructures in β-sheet were strikingly resembled to Col ff, which acted as nucleation sites for the deposition of inorganic-nanocrystals;25-26 (2) SF film supported firmly 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 β-sheet, exposing more hydrophilic groups in the outside of coatings to adsorb mineral ions to form mineralized nodules.23-24; (4) At 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 an excellent compatibility and osteogenesis in the short term evaluation (less than 1 month), the investigation of potential risk of long term in vivo still need 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.

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 diameter around 10 nm and spherical morphologies were produced by reducing Ag+ with SF, which also stabilized AgNPs to ensure uniform distribution. Due to efficiently synergetic activity of AgNPs and Gen, the PD-S-Ag/g coatings significantly inhibited bacteria growth, adhesion and formation of biofilm.


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Moreover, it evidently improved the attachment and proliferation of MC3T3 cells and simultaneously inhibited bacteria proliferation in a cell-bacteria co-culture system. The AgNPs/Gen-contained coatings supported the growth of MC3T3 cells and benefitted to 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 coatings simultaneously promoted the antibacterial activity, cellular compatibility, and osteogenic properties of orthopedic titanium, with broad application prospects.

Supporting Information

Color change of different composite solutions, corresponding core-level spectra for Ag 3d and C 1 s, the release behavior of Gen in PBS, surface observation of samples after immersion, viability of cell co-cultured with bacteria, inhibitory zones for the samples against S. aureus, the interfacial interplay between MC3T3 cells and SF-based coatings.

Acknowledgements

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

References

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Schematic illustration of AgNPs/gentamycin-embedded silk fibroin coatings with robust antibacterial and osteogenetic properties.


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Gentamicin-Embedded Silk

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