Nanosilica Hydrogel for Bone Regeneration in

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A Novel Nanosilver/Nanosilica Hydrogel for Bone Regeneration in Infected Bone Defects Shiwen Zhang, Yuchen Guo, Yuliang Dong, Yunshu Wu, Lei Cheng, Yongyue Wang, Malcolm M.Q. Xing, and Quan Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01432 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 13, 2016

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ACS Applied Materials & Interfaces

A Novel Nanosilver/Nanosilica Hydrogel for Bone Regeneration in Infected Bone Defects Shiwen Zhang1,2, Yuchen Guo1, Yuliang Dong1, Yunshu Wu1, Lei Cheng1, Yongyue Wang1, Malcolm Xing2, *, Quan Yuan1, * 1, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China 2, Department of Mechanical Engineering, Faculty of Engineering and Department of Biochemistry & Genetics, Faculty of Medicine and Manitoba Institute of Child Health, the University of Manitoba, Winnipeg, Manitoba, Canada

1 Environment ACS Paragon Plus


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ABSTRACT:

Treating bone defects in the presence of infection is a formidable clinical challenge. The use of a biomaterial with the dual function of bone regeneration and infection control is a novel therapeutic approach to this problem. In this study, we fabricated an innovative, dual-function bio-composite hydrogel containing nanosilver and nanosilica (nAg/nSiO2) particles and evaluated its characteristics using FT-IR, SEM, swelling ratio, and stiffness assays. The in vitro antibacterial analysis showed that this nAg/nSiO2 hydrogel inhibited both gram-positive and gram-negative bacteria. In addition, this non-toxic material could promote osteogenic differentiation of rat bone marrow stromal cells (BMSCs). We then created infected bone defects in rat calvaria in order to evaluate the function of the hydrogel in vivo. The hydrogel demonstrated effective antibacterial ability while promoting bone regeneration in these defects. Our results indicate that this nAg/nSiO2 hydrogel has the potential to both control infection and to promote bone healing in contaminated defects.

KEYWORDS: nanosilica; nanosilver; hydrogel; bone defects; infection


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INTRODUCTION The treatment of bone defects carries the risk of infection and treatment may even be required when infection is already present. This situation continues to be a formidable challenge for clinicians.1 Significant studies have been made regarding materials for bone regeneration and for materials with local antibiotic delivery capabilities, but there are a limited number of studies that have investigated dual-purpose biomaterials with both of these capabilities.2-3 There are currently three main types of biomaterials used as antibiotic carriers in infected bone defect restoration: inorganic biomaterials, organic biomaterials and organic-inorganic composite biomaterials.4-5 Inorganic biomaterials possess excellent bone induction qualities but have the major disadvantages of a short-lived release of the impregnated antibiotics and a resistance to further modification by other functions.6 Organic biomaterials can be tailored for slow drug release over a longer period by either chemical modification or a combination of different carriers.7 However, most organics have not been used for bone regeneration due to their weak mechanical strength and inferior bone induction capability.1 Composite biomaterials, which consist of an organic polymer and an osteoconductive mineralized component, combine the advantages of controlled antibiotic release from the organic component and the stimulation of new bone formation by the inorganic component. Unfortunately, the inorganic particles separate very easily from the organic phase and a stable composite is thus hard to achieve.8-10 New inorganic–organic composite nanomaterials have recently gained much attention due to their great potential for bone regeneration.11-12 Specific inorganic nanoparticles have been used to enhance the physical and biological properties of organic polymers with their ultra-small structures and unique physicochemical properties.13 For example, silica nanoparticles have been embedded within hydrogel networks to increase their mechanical strength,14 enhance cell adhesion,15 and induce



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osteogenesis of polymeric matrices.16-17 Moreover, silver nanoparticles have emerged as attractive antibacterial agents for medical applications.18-19 We

have

successfully

synthesized

a

biodegradable,

multifunctional

cross-linker

poly(amidoamine) (PAA) that contains disulfide bonds and agmantine. Disulfide bonds have the useful property of gradually degrading in a reducing environment. Agmatine, when combined with a carbonyl group, can mimic RGD to enhance cell adhesion.20 We then copolymerized this multi-functional cross-linker with poly(ethylene glycol) diacrylate (PEGDA) to fabricate a PEGDA-PAA hydrogel system. PEGDA based hydrogels are highly hydrophilic cross-linked polymers that have been used extensively in regenerative medicine due to their highly biocompatible swollen network structure, high porosity and proper pore size.21 We demonstrate that this organic PEGDA-PAA hydrogel has excellent biocompatibility and biodegradability, and can regulate stem cell differentiation by virtue of its tunable stiffness.16 Based on our previous study,22 we combined nanosilver and nanosilica (nAg/nSiO2) particles into this organic PEGDA-PAA hydrogel. This novel organic-inorganic nanocomposite material exhibits ideal mechanical strength as well as swelling and porous properties that are well suited for cell ingrowth. Both in vitro and in vivo tests also show that the nAg/nSiO2 hydrogel has both excellent antibacterial and osteoinductive properties. Therefore, this material has great potential for addressing the clinical challenge of treating infected bone defects.

MATERIALS AND METHODS Reagents. N,N'-Cystaminebisacrylamide (CBA) was purchased from Polysciences Inc. (Warrington,

USA),

PEGDA

(Mw=700),

Agmatine

sulfate

(AS),

N,N,N',N'-

Tetramethylethanediamine (TEMED), Ammonium persulfate (APS), Lithium hydroxide


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monohydrate (LiOH·H2O), Silver nitrate (AgNO3), Sodium borohydride (NaBH4), 3(Trimethoxysilyl) propyl methacrylate (TMSPMA), ALP staining kit, Acrylic acid, EDTA, 4% Paraformaldehyde (PFA) were all purchased from Sigma-Aldrich. Live/Dead cell staining kit was

purchased

from

Invitrogen

(Carlsbad).

The

3-(4,

5-Dimethylthiazolyl-2)-2,

5-

diphenyltetrazoliumbromide (MTT) were from Biotium Inc. (Hayward, USA). TRIzol RNA extract kit was from Ambion (Austin, USA). First Strand cDNA Synthesis kit and SYBR Green PCR Kit were from Thermo Scientific (Hudson, USA). Preparation of the hydrogels. The synthesis and TMSPMA-modification of silica nanoparticles, as well as the synthesis of poly(amidoamine) cross-linkers (PAA), were completed according to the methods previously reported by our group.22-23 Nanosilica (nSiO2) hydrogel was prepared by free radical polymerization of 6 wt% PEGDA, 0.5 wt% PAA cross-linker, 0.1 wt% acrylic acid and 2 wt% modified nanosilica using thermal dissociation of APS (0.06 wt%) as the initiator, and catalyzed by addition of TEMED at room temperature for 5 min. After gelation, nSiO2 hydrogels were equilibrated in distilled deionized water for one day and then the swollen hydrogels were equilibrated in 2 mM silver nitrate aqueous solution for another 24 h. During this time, the silver ions were exchanged from the solute to the gel networks through the free-space between the cross-linked networks, or anchored to the -COOH, –NH2, –OH groups of polymeric chains.24 Then, the silver salt-loaded hydrogels were transferred to a beaker containing 4 mM cold aqueous NaBH4 solution. The beaker was kept at 4 °C for 2 h to reduce the silver ions into silver nanoparticles. After that, the nAg/nSiO2 hydrogels were washed three times to remove unreacted silver ions and NaBH4. Hydrogel (6 wt% PEGDA, 0.5 wt% PAA and 0.1 wt% acrylic acid) and nSiO2 hydrogel served as controls without special notification.



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Characterization of the hydrogels. The morphology of the silica nanoparticle was studied using a JEM-100CX (JEOL, Japan) transmission electron microscope (TEM). And the chemical structure of the PAA crosslinker was evaluated by nuclear magnetic resonance (NMR). Fourier transform infrared (FT-IR) spectroscopy was performed to characterize the nanosilica modification and hydrogel formation. The porosity and morphology of the hydrogels were observed using SEM (Cambridge Stereoscan 120). In addition, SEM and X-ray energydispersive spectroscopy (EDS) analysis were used to characterize the presence of silver nanoparticles. EDS spectra were recorded at 20 kV. The swelling test was performed as previously described.22 Briefly, the freeze-dried hydrogels were immersed in PBS buffer. At each time point, they were removed and the surface moisture was wiped off using tissue paper. After being weighed, they were returned to the PBS buffer. The water uptake was calculated using the following formula: Water (%) = (Wt - W0)/W0 ×100 Where Wt represents the weight of hydrogels at time t and W0 is the initial weight of the dry hydrogel. The stiffness test of hydrogels was performed on a tensile Instron (5965) machine at a speed of 0.5 mm per second at room temperature. All the hydrogel samples were prepared in a self-made PDMS cylinder shape model (diameter=10 mm and height=5 mm) to make sure that they have the same shape. Before testing, all the samples were allowed to equilibrate in deionized water for 1 day. All results were obtained using triplicate samples. Antibacterial Tests. The antibacterial characteristics of the nAg/nSiO2 hydrogel were evaluated using a microplate proliferation assay as previously described.25 Antibacterial activity was also assessed through an inhibition zone test. The samples were laid on the surface of 1.5%


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agarose gel containing approximately 1 × 107 cell density of S.aureus and E.coli, followed by 24 h incubation at 37 °C. Cell culture. BMSCs were obtained from 6-8 week-old Sprague Dawley (SD) female rats. The rat femurs were aseptically removed and the bone marrow was aspirated. Then the cells were suspended and cultured at 37 0C in α-MEM (Lonza) supplemented with 10 % fetal bovine serum (Hyclone), 100 unit/ml penicillin and 100 µg/ml streptomycin (Cellgro). Unattached cells were removed after 24 h. Cells were grown to 80 % confluency, then dissociated by 0.25 % trypsin (Gibco) and subcultured used 1:3 ratios. The cells at passages 3-4 were used for our study. All the hydrogel samples for cell culture were prepared as our previous method in which the gel was formed on two glass cover slips.22 The preparation of nAg/nSiO2 hydrogel was followed by the protocol described above. Before the cells were cultured, all the gels (22 mm × 22 mm) were thoroughly washed and sterilized under UV light for 30 min. Then, the samples were put in cell culture dishes. 1 × 106 BMSCs per cm2 were seeded onto the hydrogels and incubated at 37 °C for 1 h, and then medium was added. Media was added after incubation in order to prevent cells slipping away from the hydrogel to the culture dish. This could occur if the cells and medium were added at the same time. Cell viability assay. MTT assay was used to investigate the cell viability. All the gel samples were punched into small pieces using a 6 mm diameter punch. After washing and sterilizing under UV light, the gel pieces were put into a 96-well culture plate and BMSCs were seeded. After three days of cell culture, MTT (5 mg/ml) reagent was added and further incubated for 4 h. Then the formazan salt was dissolved with 200 µl DMSO. After being thoroughly dissolved, the solution was added to a new plate and measured with a µQuant microplate reader at 570 nm (n=3). Cell viability was further assessed by Live/Dead staining. Cells on the hydrogel were



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incubated for 20 min in 2 mM calcein AM and 4 mM ethidium homodimer, then washed with PBS and imaged by an inverted fluorescence microscope (modelIX70, Olympus, Japan). Osteogenic differentiation. BMSCs seeded on gels were cultured in an osteogenic differentiation medium with 100 nM dexamethasone, 50 µM ascorbate, and 10 mM βglycerophosphate. On days 7, cells on the gels were fixed with 4% paraformaldehyde and then stained with ALP staining solution as previously described. For Real-Time PCR analysis, cells on the gels were treated with liquid nitrogen and crushed. The total RNA was extracted using TRIzol RNA extract kit. RT-PCR was carried out with an ABI 7500 qPCR system. The genespecific primers (5’–3’) were listed in table 1. Relative expression was calculated using a 2-∆∆Ct method by normalizing with GAPDH housekeeping gene expression.

Generation of infected bone defects. Female SD rats (weight ranges from 200g to 300g) were obtained from the Experimental Animal Center of Sichuan University. All the experiments were approved by the Animal Research Committee of Sichuan University and performed in accordance with international standards on animal welfare. Bone defects were created on the cranium as follows. After general anesthesia and disinfection, a midline incision was made to raise a full-thickness flap and expose the parietal bone. The periosteum was thrust aside by blunt dissection. Bilateral, 3 mm full thickness defects were made in the parietal bones with a trephine.


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During the surgery, sterile saline was applied at a rate of 2 drops per second. A resorbable collagen plug (REF 260-509-400, Bicon, USA) soaked with 107 CFU of methicillin-resistant staphylococcus aureus (MRSA) (ATCC #43300) suspended in 100 µl of sterile normal saline was then inserted into each defect. Defects filled with a collagen plug soaked with sterile normal saline were used as control. The pericranium was closed with 5-0 resorbable sutures and the skin was closed with 3-0 nylon sutures. One week after the first surgery, debridement was performed. The parietal bone was re-exposed using the same incision and all nonviable tissue was removed. During the debridement, any obvious infection was clinically verified. Then the biomaterials were inserted into each defect site. The skin was reclosed with sutures. Anti-infection evaluation. Four days after the second surgery, 2 mm × 1 mm granulation tissues were obtained from the defects for microbiological examination. The samples were then put into 10 ml of sterile normal saline and homogenized for 5 minutes. 100 µl of the solution were inoculated on an agar plate and incubated for 24 h at 37 °C. Then viable bacterial counts were taken. Meanwhile, 1 mm × 1 mm granulation tissues were taken from another defect in the three groups for RNA extraction. The total RNA was extracted using TRIzol RNA extract kit. The method for Quantitative RT-PCR was the same as above. The primers designed for each targeted gene were listed in table 1. Micro CT analysis. Four weeks after the biomaterials implantation, the parietal bones were collected and scanned in µ-CT 50 Scanner (70KV, 200 µA, 18 µm resolution). Data were analyzed with Scanco Medical Evaluation Software. The two-dimensional computed tomographic data were analyzed using Image J to determine the percentage of newly formed bone.



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Histological preparation. All samples were fixed in 4% PFA for 1 week and decalcified with 17% EDTA solution for 5 weeks before processing for paraffin sections. 5 µm coronal sections were prepared and stained with H&E and Masson staining. Statistical analysis. All values are expressed as mean ± standard deviations (SD). Significant differences between two groups are measured by one-way analysis of variance (ANOVA) followed by Tukey’s test. A P value less than 0.05 is considered to be statistically significant.

RESULTS AND DISCUSSION Preparation of the hydrogels. We first performed a surface modification of the nanosilica in order to increase its dispersibility into the PEGDA-PAA hydrogel and to synthesize a more stable inorganic-organic composite. Surface modification is an effective way to increase the hydrophobicity of the nanoparticles. This contributes to the matrix miscibility and improves the tunable interfacial interactions between the nanoparticles and the polymer.26 The most commonly used modifying agent is TMSPMA. The methoxy group of the silane coupling agent reacts with hydroxyl groups from silica nanoparticles, while the double bonds in the side chain react with the PEGDA polymer (Fig. 1A). Our novel PAA cross-linker was synthesized via Michael-addition polymerization of CBA and AS with a 1.8: 1 mole ratio and contains disulfide bonds in the backbone and guanidine in the side chains (Fig. 2B). The disulfide linkages (blue in Fig. 1B) regulate degradation of the hydrogel in a reducing environment.27 Agmatine (red in Fig. 1B), combined with an adjacent carbonyl group from acrylic acid, mimics RGD peptides in order to enhance cell adhesion. As shown in Fig. 1B, the nSiO2 hydrogel was fabricated by free radical polymerization and nAg/nSiO2 hydrogel was then prepared using NaBH4 to reduce the silver nitrate. This is a common method for nanosilver fabrication in a hydrogel network. Wang et al.28


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first introduced in situ synthesis of gold nanoparticles in a hydrogel network and this facile method has since been widely used to develop nanosilver-hydrogel composites for antibacterial applications.29-30 The reducing reaction occurs very rapidly. The nSiO2 hydrogel, loaded with silver salt, is added to the NaBH4 solution. The color of the hydrogel changes to brown, which indicate that silver nanoparticles have been generated. The resulting nAg/nSiO2 hydrogel is composed primarily of a PEGDA main chain, a PAA crosslink, nanosilica and nanosilver. Fig. 1B (right) illustrates the structure of the nAg/nSiO2 hydrogel. The PEGDA chain, PAA crosslinker and modified nanosilica form a 3D inorganic-organic network with the silver nanoparticles dispersed into the network by chemical bond adsorption.

Fig. 1. Preparation of the hydrogels. A: Schematic illustration of the TMSPMA modification of nanosilica. B: Schematic illustration of hydrogel preparation. The schematic drawing in right corner depicts the nAg/nSiO2 hydrogel structure.



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Chemical properties of the hydrogels. TEM image indicates that the size of the silica nanoparticles is about 20 nm (Fig 2A). The chemical structure of the PAA cross-linker was confirmed by 1HNMR spectra (Fig. 2B). 1H NMR (ppm): 5.6-6.4 (CH2=CH-(C=O)-), 3.4-3.6 ((C=O)NH-CH2-), 3.2-3.3 (-CH2-NH(C=NH)NH2), 2.8-2.9 (-CH2-N(CH2)2-, -CH2-SS-CH2-), 2.5-2.6 (-CH2-N(CH2)2-), 2.4-2.5 (-CH2(C=O)NH-), 1.4-1.6 (-CH2(CH2)2CH2-). The modification of the silica nanoparticles was confirmed by FT-IR (Fig. 2C, a and b). A new peak appeared at about 1730 cm-1 and is due to the C=O stretching frequency from the acrylic ester groups of TMSPMA. This suggests that the nanosilica has been successfully modified by TMSPMA. FT-IR analysis was also performed to verify the structure of the prepared hydrogels. Two new peaks appeared at 1660 cm-1 and 1544 cm-1 after conjugating the PEGDA hydrogel with PAA cross-linker (Fig. 2C, c and d). These peaks are attributed to C=O vibrations in the conjugated amide groups in the PAA cross-linker. These results indicate that the PAA crosslinker has been successfully conjugated into the hydrogels. The large amount of hydroxyl groups from the nanosilica caused a strong peak at about 3500 cm-1 (Fig. 2C (e)). The peak at 1100 cm-1 was due to the strong adsorption of Si-O-Si bond stretching and the adsorption peak at 800 cm-1 to Si-O-Si bond bending vibrations. These results collectively demonstrate that the nanosilica and PAA cross-linker have been successfully conjugated into the PEGDA hydrogel. Morphology of the hydrogels. SEM photography was employed to observe the morphology of the hydrogel and to make an assessment of its porous property. The porous property of a hydrogel plays a significant role in cell penetration and migration, tissue ingrowth and nutrient supply within the hydrogel. If the pore size is too small, it will limit cell migration towards the hydrogel center and impair nutrient and waste transportation. Conversely, pores that are too large will hinder cell adhesion and cell-to-cell inter-reaction.31 SEM (Fig. 2D, a and d) reveal that the


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pore sizes in the PEGDA-PAA hydrogel are about 100-200 µm. While the pore sizes in the nSiO2 and nAg/nSiO2 hydrogels are smaller, about 50-100 µm (Fig. 2D, b and c). This is a result of the modified nanosilica reacting with the PEGDA polymer to form a more compact network. These pore sizes are suitable for cell penetration and the transfer of oxygen, nutrients and wastes.32 Silica nanoparticles were incorporated during the construction of the inorganic-organic nanocomposite and, not surprisingly, we observed that the silica nanoparticles were inserted into the nSiO2 hydrogel network itself (Fig. 2D, e, blue arrows). In contrast, the nanosilver did not contribute to the fabrication of the main structure of nAg/nSiO2 hydrogel and is attached to the inner pore surface of the hydrogel. SEM (Fig. 2D, f, red arrows) show that many silver nanoparticles are loaded onto the pore wall surface of the nAg/nSiO2 hydrogel. However, note that it is hard to accurately judge the specific size of the silver nanoparticles from the SEM analysis.30 X-ray energy-dispersive spectroscopy (EDS) analysis was used to confirm the presence of silver nanoparticles. As shown in figure 2D (c), the absorbance band at about 3 keV reveals the presence of the silver nanoparticles. Thus, both SEM and EDS confirm that nanosilver has been successfully incorporated into the nAg/nSiO2 hydrogel. Swelling ratio of the hydrogels. The swelling behavior of a hydrogel markedly affects cell viability by influencing the transfer of nutrients.33 A higher swelling ratio results in a higher permeability for oxygen, nutrients, and other water-soluble metabolites. Our hydrogel could take up water equivalent to 6 times its dry weight (Fig. 2E). These nSiO2 and nAg/nSiO2 hydrogels exhibited even better water holding capacity during three days of observation. We attribute this ability to the inclusion of nanoparticles which act as a physical barrier to prevent the moisture from being exuded out of the hydrogel.34



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Mechanical properties of the hydrogels. Mechanical properties are another important design aspect of a biomimetic hydrogel intended for bone tissue engineering. It has been demonstrated that the stiffness of a matrix can restrict stem cell differentiation to a specific lineage.35-36 There is evidence that the stiffness of the local matrix not only has a profound influence on differentiation, it can also affect mutation and regeneration of cells.37 For example, collagencoated polyacrylamide gels with a Young’s modulus of 25–40 kPa favor osteogenic differentiation of human MSCs.35 The Young's modulus of the nSiO2 hydrogel is about 80 kPa when nanosilica is added at 2% by weight (Fig. 2F). This is significantly higher than the 32 kPa modulus of the hydrogel alone. The TMSPMA modified silica nanoparticles, which have carbon double bonds, can react with the PEGDA polymer and form a strong inorganic-organic hydrogel network. This mechanical strength ensures that the hydrogel retains its initial shape at the implant sites. The stiffness of nAg/nSiO2 hydrogel is slightly, but not significantly, higher than nSiO2 hydrogel. This is because the nanosilver does not contribute to fabrication of the main structure of the nAg/nSiO2 hydrogel.


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Fig. 2. Characterization of the hydrogels. A: TEM images of the synthesized silica nanoparticle. Scale bar=20nm. B: 1H NMR spectra of PAA cross-linker. C: FT-IR spectrum of the nSiO2 (a); TMSPMA modified nSiO2 (b); PEGDA hydrogel (c); PEGDA-PAA hydrogel (d); nAg/nSiO2



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hydrogel (e). D: SEM showing the structure of the hydrogels. c: top right corner is EDS spectrum of the nAg/nSiO2 hydrogel; d: a higher magnification of the PEGDA-PAA hydrogel; e: a higher magnification of the nSiO2 hydrogel (blue arrows indicate the silica nanoparticles); f: a lower (the small picture) and higher magnification of the nAg/nSiO2 hydrogel (red arrows indicate silver nanoparticles). (a, b, c scale bar=100 µm; d=50 µm; e=20 µm; f=1 µm). E: Swelling ratio of the hydrogels. F: Stiffness of the hydrogels. "*" means p

Nanosilica Hydrogel for Bone Regeneration in

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