Facile and highly efficient approach for the fabrication of multifunctional silk nanofibers containing hydroxyapatite and silver nanoparticles Faheem A. Sheikh,1,2 Hyung Woo Ju,1 Bo Mi Moon,1 Hyun Jung Park,1 Jung-Ho Kim,1 Ok Joo Lee,1 Chan Hum Park1,3 1

Nano-Bio Regenerative Medical Institute, College of Medicine, Hallym University, Chuncheon, 200-702, South Korea Department of Chemistry, University of Texas-Pan American, Edinburg, Texas 78539 3 Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Hallym University, Chuncheon, 200-702, South Korea 2

Received 2 September 2013; revised 11 October 2013; accepted 22 October 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35024 Abstract: In this study, a good combination consisting of electrospun silk fibroin nanofibers incorporated with highpurity hydroxyapatite (HAp) nanoparticles (NPs) and silver NPs is introduced as antimicrobial for tissue engineering applications. The variable pressure field emission scanning electron microscope results confirmed randomly placed nanofibers are produced with highly dispersed HAp and silver NPs in nanofibers after electrospinning. The X-ray diffraction results demonstrated crystalline features of each of the three components used for electrospinning. Moreover, the TEM-EDS analysis confirmed the presence and chemical nature of each component over individual silk nanofiber. The FT-IR analyses was used confirm the different vibration modes caused due to functional groups present in silk fibroin, Hap, and silver NPs. The obtained nanofibers were checked for antimicrobial activity by using two model organisms Escherichia coli and Staphylococcus aureus. Subse-

quently, the antimicrobial tests have indicated that prepared nanofibers do possess good bactericidal activity. The ability of N,N-dimethylformamide and silk fibroin used to reduce silver nitrate into silver metal was evaluated using MTT assay. The nanofibers were grown in presence of NIH 3T3 fibroblasts, which revealed toxic behavior to fibroblasts at higher concentrations of silver nitrate used in this study. Furthermore, cell attachment studies on nanofibers for 3 and 12 days of incubation time were minutely observed and correlated with the results of MTT assay. The reported results confirmed the high amounts of silver nitrate can lead to toxic effects on viability of fibroblasts and had bad effect in cell C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part attachment. V A: 00A:000–000, 2013.

Key Words: antimicrobials, nanofibers, silk, biomaterials, biocompatible, cell viability, cytotoxicity, cell attachment

How to cite this article: Sheikh FA, Ju HW, Moon BM, Park HJ, Kim J-H, Lee OJ, Park CH. 2013. Facile and highly efficient approach for the fabrication of multifunctional silk nanofibers containing hydroxyapatite and silver nanoparticles. J Biomed Mater Res Part A 2013:00A:000–000.

INTRODUCTION

Formation of super-bugs has surfaced as major problem because of the frequent use of antibiotics by humans. Basically, the genetic transformations in microbial strains result to show resistance toward antibiotics and which in turn leads to nonfunctioning of available antibiotics.1 Therefore, continuous and tremendous steps are considered to improve the quality of antibiotics so as they are effective against transformed microbes. To have complete removal of pathogenic strains, there is a need to have an alternate strategy, rather than using organic antibiotics locally at the site of operation. Silver and its forms are considered as natural inorganic antibiotics. They had been used since ancient

times for the purpose of wound healing. When silver comes in contact with the microorganisms, immediately it leads to distortion of cell wall, which later leads to death of these organisms. Therefore, progressive steps are made for future use of silver-based materials in present technological world.2 Chances to develop minimum resistance and/or broad-spectrum activities against both Gram-negative and Gram-positive bacterial strains are its attractive features. Furthermore, immediate death of microorganisms upon contact with the silver ions can lead them to replace nonpotent antibiotics. The mechanism for development of minimal resistance and/or no resistance in microorganisms against silver has been well documented.1,3 In addition, having

Correspondence to: C. H. Park; e-mail: [email protected] Contract grant sponsor: Hallym University Research Fund and a grant from Bio-industry Technology Development; contract grant numbers: 112007-05-2-SB010 and 111100-03-3-SB010 Contract grant sponsor: Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea

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antimicrobial properties of silver, it had been found that silver-based dressing materials can promote wound healing and reduce scar appearance in a dose-dependent manner in clean wounds.4 Silk fibroin obtained from cocoons of silkworms which had a long history of applications for using in sutures. Its beneficial peptides composed of RGD sequences that can promote cell adhesion, migration, and proliferation.5,6 The attractive properties of silk fibroin to be helpful in promoting cell differentiation are useful for selecting them as a material of choice for tissue engineering applications.5 Furthermore, efficient biocompatibility, minimal inflammatory response to host tissue, relatively slow biodegradation rates compared with other materials, and easy availability from sericulture industry make the silk fibroin a desirable candidate for various astonishing applications.7 On the other hand, hydroxyapatite (HAp) is considered as major solid component of the human bone and teeth. It can be used as a vital implant because of its excellent biocompatibility, bioactivity, nonimmunogenicity, noninflammatory behavior, and osteoconductive nature.8 The desirable mechanical support to sustain pressure associated from surrounding host tissues are the vital assignments of HAp in living tissues. However, once isolated and purified, its loose and particulate nature makes it really hard to exploit in making of tissue scaffolds.9 To overcome this barrier, a suitable polymer as blend material is selected to facilitate proper cell seeding and diffusion of nutrients for healthy growth of cells during the initial period of implantation, which is considered as crucial.10 An interesting strategy using nanotechnology to formulate natural and synthetic polymers into tissue scaffolds by the aid of electrospinning technique is now industrialized.11 Historically, it was first described in patents of Cooley12 in the early 1900s. However, first reports on using this technique to obtain cellulose acetate, cellulose propionate, and wool–cellulose acetate fibers were published in the 1930s.13 In a typical electrospinning process, polymer solution is placed in a syringe, which is connected to a high power supply, capable of generating high voltage difference (5 to 30 kV) between the syringe needle and a grounded collector. As the polymer solution is exposed on tip of needle, the electrical charges on the polymer solution promotes its stretching, which eventually forms ultrafine fiber. During this process, the solvent associated with polymer evaporates immediately and forms a dry polymer fiber that travels in a chaotic pattern and gets deposited on the grounded collector.14 Nanofibers received from this technique have drawn considerable attention because of their small diameter and particularly a web-like structure, which mimic the topology of extracellular matrix existing in human body.15 For using nanofibers as implant and simultaneously have wound healing ability, it should have the following characters: good mechanical properties, water insolubility, appropriate pore size, and noninvasiveness in a sense that it will favor the epithelialization. Strategies to improve growth of bony tissues by using the HAp NPs had been frequently used in modifying the polymeric nanofibers. This includes either by

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partial attachment of HAp NPs on nanofibers surfaces by incubating them in the stimulated body fluid16 or directly blending the polymer solution with HAp NPs and then electrospinning these solutions into nanofibers.17 Nevertheless, the nanofibers do have solitary importance, but also the polymer matrix plays a distinct role; hence, many polymers have been exploited. For instance, the polymeric nanofibers of cellulose acetate, poly(acrylonitrile), poly(caprolactone), poly(methyl methacrylate), poly(vinyl alcohol), and polyimide had been modified using silver NPs, so they will represent antimicrobial properties, which in future will aid in wound healing.18–23 Unfortunately, all these reports have focused only on the synthesizing procedure and ignored about cell toxicity by using the final products in tissue engineering. An efficient and multicomponent combination design in which silk fibroin HAp and silver NPs fabricated into nanofibers will be a breakthrough to focus the different issues related with advanced antimicrobial activity and nontoxic behavior of implants. It is noteworthy to mention that there are only few reports indicating the use of both HAp and silver together to modify nanofibers. For instance, series of investigations were reported by Nirmala et al.24,25 mentioning use of synthetic polymer such as poly(caprolactone) modified with HAp and Ag NPs. However, there is no such report mentioning the use of natural polymers such as silk fibroin in the form of nanofibers, which is imparted with properties of HAp and silver NPs to tissue engineering applications. In this work, a good combination consisting of silk/HAp and silver nitrate nanofibers was prepared by using electrospinning for potential antimicrobial activities. Moreover, evaluation of the obtained nanofiber matrices for morphological properties and crystalline structure had been investigated. Antimicrobial activities of nanofibers were investigated using two model microorganisms. In addition, the prepared nanofibers were tested for cell cytotoxicity using fibroblasts. Accordingly, the obtained results indicated these nanofibers matrix could be properly used as recommended candidate for many implant applications. EXPERIMENTAL SECTION

Materials Cocoons from Bombyx mori were kindly gifted by Rural Development Administration (Suwon, Republic of Korea). Rod-shaped HAp NPs measuring 30–60 nm were purchased from (DaeJung, Siheung, Gyeonggi, Korea). Poly(ethylene oxide) (PEO) with an average molecular weight of 200,000 used as sacrificial polymer to give appropriate bending stability to electrospun silk solutions. N,N-dimethylformamide (DMF) (analytical grade), phosphate buffer saline (PBS) 0.1M, pH 7.4, dimethyl sulfoxide (DMSO) (99.9%), and silver nitrate (AgNO3) were purchased from (Sigma-Aldrich, St. Louis, MO). NIH 3T3 fibroblast cells were purchased from ATCC (Manassas, VA). Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, cocktail of 1% penicillin–streptomycin, Trypsin, were obtained from Welgene, Fresh MediaTM (Dalseogu, Daegu, Korea). Trypan Blue Stain 0.4% was obtained from GibcoV (Life R

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ORIGINAL ARTICLE

Technologies Corporation, Gaithersburg, MD). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent used to check the cell viability was purchased from (Duchefa Biochemie, Haarlem, The Netherlands). Tissue culture flasks and microplates for cell seeding and growth were purchased from BD FalconTM (Winston-Salem, NC) and SPL Life Sciences (Pocheon-si, Gyeonggi-do, Korea). DifcoTM LB Broth, Miller (Luria-Bertani) and BactoTM Agar used to culture microbial strains were purchased from Becton (Dickinson & Co, Sparks, MD). For checking antimicrobial activity, two microbial strains, Escherichia coli (ATCC 43890) and Staphylococcus aureus, obtained from (National Veterinary Quarantine service Korea) were used as a model organisms to check antimicrobial activity. Characterization Prepared nanofibrous membranes decorated with HAp and silver NPs were characterized with various microscopic and spectroscopic techniques. The variable pressure field emission scanning electron microscope (VP-FE-SEM) EVOV LS10 at the Korean Basic Science Institute, Chuncheon, equipped with energy-dispersive X-ray spectroscopy (EDS) obtained from (Carl Zeiss SMT, Oberkochen, Germany) was used to investigate the morphology and elemental detection of nanofibers. The samples were loaded on aluminum stubs, pasted on double-side carbon tape, and sputter-coated using a thin layer of gold-palladium for 120 s for two consecutive cycles at 40–45 mA with the Ion Sputter 1010 (Hitachi, Chiyoda-ku, Japan). The micrographs from each sample were taken at an accelerating voltage of 3 kV and with magnifications of 30K. The EDS images were captured at an accelerating voltage of 10 kV and with magnifications of 60K. Transmission electron microscopy (TEM) equipped with EDS was done by JEOL JEM-2200FS operating at 200 kV (JEOL, Akishima-shi, Japan). For the TEM-EDS analysis, samples were prepared by dispersing 10 mg of nanofibers in 200 lL of ethanol and subsequently dispersing by bath sonicator using locally supplied ultrasonic cleaner (60 kHz, Shenzhen Codyson Electrical Co., Shenzhen, Guangdong, China) for 120 s. After dispersing the nanofibers, 20 lL of dispersion was pipetted out by micropipette and carefully poured on 200 mesh copper grid. The extra solution was removed using Kimwipes supplied by (Kimberly-Clark Professional, GA), and the grid was allowed to dry overnight at room temperature and examined under microscope. Information about the phases and crystallinity was obtained using PANalytical diffractometer (HR-XRD, X’pert-pro MPD, Almelo, The Netherlands) with Cu, Cr (k 5 1.540 A) radiation over Bragg angle ranging from 20 to 80 . Vibrational modes occurred due to functional groups in nanofibers; the Fourier transform infrared spectroscopy (FT-IR) analysis was done using BIORAD (Cambridge, MA). The samples were directly loaded on ATR window, and spectra’s were collected using Excaliber Series by averaging 32 scans with the resolution of 4 cm21. For checking the cell attachment on nanofibers by VP-FE-SEM, the images were captured with an accelerating voltage of 3 kV with magnifications of 5K. The zones of inhibition on microbial plates were studied

FIGURE 1. 11.Schematic illustration showing the design of electrospinning setup for fabricating the nanofibers. [Color figure can be viewed in the online issue, which is available at wileyonline library.com.]

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by taking images using Molecular ImagerV ChemiDocTM XRS System-BIORAD (Seoul Korea). R

Isolation of silk fibroin from cocoons A stepwise, methodology as described in our previous publication was adopted to obtain sericin free fibroin solution from silk cocoons, which later on were used for electrospinning.26 First, clean and insect free cocoons from B. mori were selected and chopped into suitable pieces to boil in 0.02M Na2CO3 for an hour. Subsequently, these cocoons were washed with deionized water (two to three times) to remove the unbound sericin. Later on, the samples were dried at room temperature for 1 day in clean bench. Furthermore, 60 g of degummed silk was dissolved in ternary solvent composed of CaCl2/Ethanol/H2O (32/26/42, wt/wt/ wt) at 98 C for 40 min in round-bottomed flasks. Resulting protein mixture was filtered through miracloth (Calbiochem, San Diego, CA) to remove small aggregates. Furthermore, this solution was dialyzed against deionized water using a dialysis tubing with molecular weight cutoff 12,000 to 14,000 Da (Spectra/PorV, Rancho Dominguez, CA) for 3 days, in sufficient amount of deionized water, which was exchanged once a day. The final aqueous silk fibroin solution was calculated to be 8 wt % on dry weight bases. The resulting aqueous silk fibroin solutions were stored in a refrigerator to avoid any denaturation and were used before 15 days of time for electrospinning. R

Polymeric solution preparation for electrospinning The polymeric solution containing HAp NPs were prepared using 20 mL of 8 wt % of aqueous silk fibroin solution. The 4 mL of previously prepared 30 wt % PEO solution was added as a “sacrificial” polymer to maintain appropriate viscosity, so as to have proper bending instability for fiber formation, during the electrospinning process. As indicated in (Fig. 1), this solution was mixed together with silk solution and loaded in syringe 1. On another hand, PEO/HAp colloidal solution was prepared by adding 2 g of PEO in 20 mL of

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remove sacrificial PEO from nanofibers and at the same time lead to crystallization of nanofibers. Furthermore, these samples were freeze-dried.

0.001 molar solution of phosphate buffer saline (PBS), and this solution was mixed well to solubilize. To this solution, HAp NPs were added to give the final concentration of 50% HAp with respect to 8% of aqueous silk fibroin solution. The HAp NPs were agitated using an ultrahigh sonication device using Sonics Vibra-cell model (VCX 750, Newtown, CT), operating at 20 kHz with amplitude of 20% for a period of 1 min. These samples were viewed as homogeneously dispersed and well stabilized without being settled at the bottom after sonication. Furthermore, these dispersed HAp/PEO solutions were filled into syringe 2 to be used for electrospinning. For electrospinning of silk-HAp nanofibers containing silver nitrate, 0.5, 1.0, and 1.5% of silver nitrate with respect to silk solution was dissolved in 1 mL DMF and incubated overnight in a shaking incubator. It is noteworthy in mentioning that, the reduction of silver nitrate to silver metal had been accompanied using N,N-dimethylformamide (DMF) as reducing agent.27 The obtained silver nitrate solutions in DMF appeared darker in color depending on the concentration of silver dispersions. Finally, these dispersions were mixed with the contents of syringe 2.

Zones of inhibition Analysis of zones of inhibition (ZoI) was performed by making samples from silver-free nanofiber mats and silver containing ones. The samples were cut into round disk shape by biopsy puncher having diameter of 6 mm. The samples were sterilized by UV light for 5 min. The two microbial strains E. coli and S. aureus were efficiently raised and spectroscopically checked to reach density of 1 3 104 cells/mL by McFarland method. The test has been performed as follows: a cell suspension of 100 mL of 1 3 104 cells/mL was inoculated over a plate containing solidified nutrient agar medium onto disposable sterilized petriplates by spread plate method. Immediately after spreading of microbial strains, sterilized nanofiber samples were gently placed over the solidified agar gel under aseptic conditions. Plates were incubated for 12 h at 37 C, and then zones of inhibition were observed.

Electrospinning process The electrospinning of silk-HAp nanofibers was achieved using our three-way electrospinning procedure.26 The electrospinning instrument used in fabrication of nanofibers was obtained from (eS-robotV, ESR-200R2D, NanoNC, Geumcheon-gu, Seoul, Korea). The three-way connector exploited to mix Silk1PEO solution and PEO1HAp1silver nitrate solutions for a short time before it gets precipitated because o the bond formation with HAp and silk fibroin.26 In this regard, Figure 1 presents the schematic illustration of the electrospinning technique in this study. As described in scheme, from syringe 1, the Silk1PEO solution is supplied to one end opening of three-way connector, and from syringe 2, the solution containing PEO1HAp1silver nitrate was supplied to another end opening. Finally, configuration of stopcock three-way connector allows the two solutions to blend properly and eventually helps to flow forward to the needle tip because of the continuous pressure applied from syringe pump. The solutions for nanofibers were injected using 10 mL disposable plastic syringes fitted with a 13needle gauge (2.41 mm OD31.80 mm ID). The syringes were mounted on an adjustable stand, and flow rate of 0.4 mL/min was adjusted using a multi-syringe pump to keep the solution at the tip of the needle and simultaneously be sure that excessive solution does not drip off. A high-voltage power supply instrument capable of generating power supply of (120 kV) was connected to tip of needle with an alligator clip helping to eject out the fibers and negative electrode (cathode) with an applied voltage of (21 kV) was attached to the flat bed metallic collector. Solutions were loaded in four syringes and were mounted in the parallel plate geometry at 45 down-tilted from the horizontal baseline. The working distance between needle tip and collector was kept 12 to 15 cm. The as-spun nanofibers were treated by incubating the samples in 100, 75, 50, and 0% of ethanol in deionized water for 10 min. This helped to

Cell viability and cell attachment studies NIH 3T3 cells stored in liquid nitrogen tank were taken out and incubated at 37 C for 3 min to form semisolid suspension of cells. The suspension from ampules was taken out and added with fresh media, centrifuged, enriched with fresh media, and allowed to incubate at 37 C for 3 days to complete first subculture. In this study, cells were used after two subcultures to check the cell viability and cell attachment with renewal of media after every 2 days of time. The nanofibers used for cell studies were pierced into disk shapes using biopsy punchers (Kasco, Keys Cutaneous Punch, Sialkot, Pakistan) forming 6 mm round disks, giving it an appropriate diameter to fit in a 96-well plate. Each nanofiber disk was sterilized by dipping it in 70% ethanol in 6-well plate for 30 min. The excess of ethanol on nanofibers after sterilization was rinsed by dipping the samples in 5 mL of PBS (two times) and final dip in 5 mL of DMEM. Furthermore, the samples were transferred on 96-well plates in triplicates. A 100 lL of cell suspension containing 25,000 cells/mL was counted using cell counting method and was carefully seeded on top of sterilized nanofiber disks in the 96-well plate. These plates were incubated at 37 C for 30 min to allow cell adhesion. Afterward, 100 lL of fresh medium was added in each well, and the plates were incubated in a humidified incubator with 5% CO2 environment at 37 C for 1, 2, and 3 days. After culturing the fibroblasts in presence of nanofibers, the cell viability was evaluated by using MTT reduction assay. In brief, the media from 96-well were suctioned out and treated with 200 lL of MTT solution, by mixing the contents by side-tapping, and then these plates were incubated at 37 C for 2 h. In these wells, MTT solution was trashed out after incubation and added with 200 lL of DMSO and subsequently rocked to form purplish bluecolored formazan solution. The solubilized formazan appearing from each well was transferred to fresh wells of 96-well

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FIGURE 2. Variable pressure field emission scanning electron microscopy (VP-FE-SEM) results of the nanofibers mats after crystallization and subsequent crystallization. The morphology of pristine silk-HAp-Ag-0% nanofibers (A), silk-HAp-Ag-0.5% (B), silk-HAp-Ag-1.0% (C), and silk-HApAg-1.5% (D).

plate for spectrophotometric analysis at 540 nm in an ELISA microplate reader (Molecular Devices, SpectraMaxV Plus 384, Sunnyvale, CA). The cell viability was obtained by comparing the absorbance of cells cultured on the nanofiber scaffolds with that of the control well containing DMSO. For cell checking attachment on nanofibers, the cells were allowed to grow for 3 and 12 days’ time. For this purpose, the cell fixation and dehydration was achieved by rinsing the samples twice with PBS followed by fixation with a 3 vol % glutaraldehyde solution for 4 h. After cell fixation, the samples were rinsed with PBS and then dehydrated with graded concentrations of ethanol (20, 30, 40, 50, 75, and 100 vol % ethanol) for 10 min each. Finally, the samples were kept overnight in a vacuum oven and observed in VPFE-SEM to determine cell attachment. R

RESULTS AND DISCUSSIONS

Figure 2 shows the VP-FE-SEM results for the nanofibers after the electrospinning processes. More precisely, Figure 2(A) shows the morphology of silk HAp nanofibers. In this figure, we can see the nanofibers are defect and bead-free. Moreover, Figure 2(B–D) shows the morphology of nanofibers modified with silver nitrate. From these figures, it can be seen that general morphology of nanofibers had not been changed by the introduction of silver nitrate. However, it can be observed that there is an increase in diameters of nanofibers upon the addition of silver nitrate. Moreover, an important observation can be made after looking at Figure. 2(C and D), that the presence of nanoparticles (NPs) on nanofiber surfaces is obvious. The reason why these NPs appeared outside nanofibers can be co-related with changes

FIGURE 3. Variable pressure field emission scanning electron microscopy (VP-FE-SEM) equipped with EDS results for nanofibers. EDS spectra for pristine silk-HAp nanofibers (A) and spectra for silk-HAp nanofibers modified with silver nitrate (B). The in-set figures represent the area under investigation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 4. Transmission electron microscope (TEM) results for the morphology of pristine nanofibers (silk-HAp-Ag-0%). The top in-set represents the high magnification of the area indicating presence of HAp NPs in nanofibers and bottom in-set represents the HR-TEM of the en-circled area indicated by arrow.

that occurred in viscosity of solutions due to addition of silver nitrate used for electrospinning.28 Basically, because of increase in conductivity which leads to form smaller diameter results nonaccumulation of HAp NPs in polymer solution. This further leads in landing of NPs outside nanofibers surfaces. Figure 3 shows the results of EDS analysis of the pristine and one of the nanofibers modified with silver nitrate. Figure 3(A) represents the data originating from pristine nanofibers. The inset image of this figure indicates the area selected for point EDS analyses. The spectra in Figure 3(A) shows that the elements present are Ca, P, N, C, and O,

FIGURE 5. Transmission electron microscope (TEM) results for the morphology of nanofibers modified with silver nitrate (silk-HAp-Ag0.5%). The top in-set shows the HR-TEM images of HAp NP and bottom in-set indicates the presence and location of Silver NP.

which further confirms the nanofibers are composed of both protein and HAp. Moreover, the data for nanofibers modified with silver nitrate is presented in Fig. 3(B). From this figure, it can be seen that in addition to the peaks responsible for confirming the presence of silk protein and HAp, it also contains peaks responsible for silver which further proves the successful completion of electrospinning along the silver nitrate. Because of the poor resolution of VP-FE-SEM, it is hard to differentiate the internal contents of any material. However, TEM with its high resolution can easily help to differentiate between the internal contents of any compound. Therefore, we invoked TEM examinations to reveal the internal contents of nanofibers. In this regard, Figure 4

FIGURE 6. Transmission electron microscope (TEM) equipped with EDS results for the pristine silk-HAp nanofibers. Figure representing the morphology of nanofiber and its encircled area under investigation for HAp NP (A). The line EDS spectra for the nanofiber under investigation. Underneath in-set figures represents the elements detected in (red) are for Ca and in (cyan) are for P (B). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 7. Transmission electron microscope (TEM) equipped with EDS results for the nanofibers modified with silver nitrate. Figure representing the morphology of nanofiber and location of HAp and silver NPs (A). The line EDS spectra for the nanofiber under investigation. In-sets figures represents the elements detected in (blue) are for Ca, (green) are for P and in (red) are for Ag (B). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

shows the TEM image of silk/HAp nanofibers which shows the presence of HAp NP inside the nanofibers, which was not possible to view with VP-FE-SEM. Moreover, its top inset figure shows the high-magnification image of the encircled area, which clears understanding about the rod-shaped morphology of HAp NPs used to blend with PEO solution. The bottom inset image in this figure indicates the HR-TEM image of HAp NP. From this image, apparent crystal pattern of NPs can be seen without any dislocation and/or imperfection in the lattice pattern of Hap.29,30 Figure 5 represents the TEM results of one of nanofibers modified with silver nitrate. In the top inset of this figure, which shows the highmagnification image of the encircled area (Fig. 5), we can see the clear presence of silver NPs. It is observed that silver NPs are spherical in shape and are having diameter of around 5–6 nm. Moreover, the bottom inset figure shows the high-magnification image of the encircled area hypotheses to be HAp NPs. In this regard, the bottom inset image shows the morphology of HAp NP which is similar with that of the HAp NPs present in that of silk-HAp nanofibers. Over-

FIGURE 8. The XRD results of the nanofibers at 2h values from 20 to 80 .

all, from both the inset images, it clearly reveals the presence of HAp and silver NPs, which could not be seen while using VP-FE-SEM. To have a clear picture about the chemical nature of the artifacts present in nanofibers in more precise manner, TEM equipped with EDS analyses was invoked. In this regard, Figure 6 shows the TEM-EDS results of the silk-HAp nanofiber. The area under investigation for chemical nature of HAp selected for characterization is presented in Figure 6(A). Figure 6(B) shows the results of line mapping; as indicated, the spectra originating from mapping analyses in this figure show the presence of overlapped two peaks at the location hypothesized as HAp NPs. The spectra at bottom insets reveal overlapped two peaks in cyan codes for phosphorous and in red is coding for calcium, which is the composite nature of HAp. Figure 7 shows the TEM-EDS results for one of the silk-HAp nanofibers modified with silver nitrate. In this image, [Fig. 7(A)], we can see in addition to HAp NPs, nanofibers do contain silver NPs which are centrally located. The results of the line mapping of the nanofibers covering HAp and silver NPs is presented in Figure 7(B). We can clearly see the line mapping gives us spectra in blue and green indicating the presence of calcium and phosphorous, which corresponds to composition of HAp NPs and the spectra expressed red in color indicates the presence of silver NPs. From these results, it clearly demonstrates the location and chemistry of the compounds present in silk-HAp nanofibers containing silver NPs. X-ray diffraction (XRD) can be used as highly stable technique to investigate the crystalline nature of any material. Figure 8 shows XRD data for the nanofibers facilitated by using stopcock connector to support the immediate mixing of aqueous silk/PEO solution and silver nitrate/HAp/ PEO colloids. In this figure, nanofibers modified with HAp NPs show various diffraction peaks at 2h values of 31.77, 32.90, 34.08, 40.45, and 46.71 that correspond to the crystal planes of (211), (300), (202), (310), and (222), which is

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FIGURE 9. The FT-IR spectra of the nanofibers obtained after electrospinning and freeze drying. Pristine silk-HAp-Ag-0% (spectrum A), silk-HApAg-0.5% (spectrum B), silk-HAp-Ag-1.0% (spectrum C), and silk-HAp-Ag-1.5% (spectrum D).

in proper agreement with (JCPDS, data base).31 The diffraction peaks at 2h values of 38.11, 44.27, 64.42, and 77.47 corresponding to (111), (200), (220), and (311) crystal planes implies the persistence of crystalline silver NPs.32 This indicates that the obtained nanofiber mats from silk/ silver nitrate colloids do have HAp and silver NPs, which simultaneously confirm the ability of DMF as reducing agent.27 Moreover, it can also be observed from this figure that, the intensity at (111) main plane in the standard silver crystal lattice (at 2h values of 38.11 ) increases with increasing the silver nitrate content in the original colloid which leads to more silver NPs in the electrospun nanofiber mats. Changes occurred in silk fibroin nanofiber composition because of the incorporation of HAp and silver nitrates were evident from FTIR spectra. Figure 9 depicts FTIR spectra from 500 to 2000 cm21 for Silk-HAp-Ag-0% nanofibers (curve A), Silk-HAp-Ag-0.5% nanofibers (curve B), Silk-HApAg-1.0% nanofibers (curve C), and Silk-HAp-Ag-1.5% nanofibers (curve D). It is clear that Silk-HAp-Ag-0% nanofibers combination carries the peaks responsible for silk and HAp NPs. For instance, the peaks appeared at 1621, 1517, and 1447 cm21 are due to vibration caused in (amide I), (amide II), and (amide III) regions of the silk fibroin. It was revealed that with the addition of silver nitrate, the peak for the (amide I), (amide II), and (amide III) band shifted toward higher wavenumbers. The displacement number was about 2, 3, and 2 units, which indicates the strong interaction between silk fibroin and silver ions, and it also puts light on transformation from random coil to b plated sheath structure in silk fibroin nanofibers. The vibration modes caused due to presence of Ca and P attributing to the presence of HAp NPs are also expressed in FT-IR results. In this

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regard, small shoulder peaks at 1449 cm21 is occurring due to existence of a Ca-O phase in nanofibers. The strong intensity peaks appearing at 1028 and 1098 cm21 can be attributed due to asymmetric stretching mode vibration in PO423 and a medium intensity band at about 961 and 843 cm21 result from P-O asymmetric stretching of stretching vibrations of PO423.33 In addition, a sharp peak at 841 cm21 is assigned to the OAH bending deformation mode in the nanofibers. Moreover, it can be observed a finger print region for HAp NPs, shows sharpening of peaks upon addition of silver nitrate. Possibly, it can be due to formation of chemical bond between silver nitrate and HAp NPs. The vibrations caused due to co-electrospinning of silver nitrate during electrospinning process in the nanofibers can also be seen. Because the process designed in our experiments was to reduce the silver nitrate to silver metal by using reducing capabilities of DMF and also to take advantage of the silk protein as natural reducing agents. It was hoped that FT-IR would put some light on whether the conversion of nitrate was completed or not. For instance, the peaks occurring due to the presence of nitrate groups can be seen at 1342 cm21 at this region; this asymmetric vibration is due to presence of nitrate group which cannot be completely reduced at higher concentrations of AgNO3. It is noteworthy to mention, that in case of nanofibers with higher concentration of silver nitrate reduction, process is not completed, either by action of DMF and/or by interaction of silk fibroin at a time of mixing by three-way stop connector. Reasons can be less amount of DMF (i.e., 1 mL for 12 h) used and the short time interaction of silk fibroin with silver nitrate (i.e., 3–4 h times during the electrospinning process). Historically, silver is considered as material of choice as far as antimicrobial agents are considered. It is also

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FIGURE 10. Results of the zones of inhibition after culturing the Escherichia coli and Staphylococcus aureus for 12 h of incubation.

established that silver enhances the epithelization in experimental animals.4–34 Pure silver and its forms are potent antimicrobials against various bacterial species. The exact mechanism how silver participates in distortion of bacteria is unfortunately still unclear. However, it is believed that sliver mainly takes part in denaturation and oxidization for cell organelles, which lead to suppress the cell division, and later on leads death of microbes. It is well established, that when a critical amount of antibacterial compound (capable of inhibiting microbial growth) is in contact with bacterial lawn the clear area near that contact is formed and these areas are referred as zones of inhibition (ZoI). Figure 10 shows the 12 h incubated petriplates of E. coli and S. aureus grown in presence of circular nanofiber disks. From this figure, we can clearly observe the ZoI around the circular nanofiber disks containing different percentages of silver (i.e., 0.5, 1.0, and 1.5%) are visible, whereas in case of silkHAp nanofibers (0%) nanofibers, no ZoI are present. Formation of ZoI around the nanofiber disks is an indicator for inhibition of bacterial colonies near the vicinity of nanofiber disk edges. Basically, because of presence of silver in the nanofiber disk, it may lead to death of the microorganism around the disk, which results in formation of clear circular zone (i.e., ZoI). It is also considered that diameter of the inhibited area is a measure of the effectiveness or resistance of the bacteria toward organic antimicrobial agent. However, in case silver a previous work has concluded that zone of inhibition is only qualitative and provides no means of assessing quantities of silver that have been released into agar medium.35 Therefore, diameter of a zone is not in itself sufficient to determine efficacy of silver and its forms. Accordingly, we did not find any significant difference with varying the silver content in nanofiber mats. Silver is considered as materials of choice from long time as far as antimicrobial agents are considered. It is also well established that silver enhances the epithelization.4 It is noteworthy in mentioning, that the nitrates present in silver nitrate are toxic to human body and can cause blue and/or bluish-grey colored pigmentation, which is termed

as argyria.36 To find out nitrate content of AgNO3 is completely reduced out by action of DMF and/or incubation in presence of silk fibroin during electrospinning. This will simultaneously address the degree of toxicity with the presented methodology. Therefore, we cultured human fibroblasts in presence of the nanofiber mats. At this stage, to understand the effect of the prepared nanofibers on cell viability, nanofibers were seeded with NIH 3T3 fibroblasts and results of cell viability are displayed in Figure 11. From these results, it was observed that cell growth was improved until the (0.5%) of silver nitrate was present in nanofibers. In other words, 1.0 and 1.5% of silver nitrate in nanofibers show toxic behavior on the growth of cells. The rationale may be that reduction method in our system can reduce the nitrate up to its maximum levels (i.e., 0.5%). Beyond this point, both DMF and fibroin proteins fall short to reduce large nitrate groups present in final electrospinning solution; therefore, higher concentrations show toxic behavior. Furthermore, to corroborate the findings of cell viability, the morphological appearance of cells attached on nanofiber mats was investigated after 3 and 12 days of culturing

FIGURE 11. Results of cell viability using MTT assay after culturing the NIH 3T3 fibroblasts for 1, 2, and 3 days.

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FIGURE 12. VP-FE-SEM results after cell fixation. The pattern of cell attachment on surfaces after culturing for 3 days; silk-HAp-Ag-0% nanofibers (A), silk-HAp-Ag-0.5% (B), silk-HAp-Ag-1.0% (C), and silk-HAp-Ag-1.5% (D). The pattern of cell attachment on surfaces after culturing for 12 days; silk-HAp-Ag-0% nanofibers (E), silk-HAp-Ag-0.5% (F), silk-HAp-Ag-1.0% (G), and silk-HAp-Ag-1.5% (H).

fibroblasts as photographed in Figure 12. From this figure, one can clearly visualize the cell attachment and cell spreading in the nanofiber matrix. This phenomenon of cell growth on nanofiber membranes exactly matches with results of cell viability tests. In addition, the observed morphologies in all the nanofiber compositions clearly support that pristine and nanofibers containing 0.5% silver nitrate shows advanced cell attachments, compared with that of nanofibers with higher contents of silver nitrates. Furthermore, the results of cell cytotoxicity provide decidable permission to use the prepared nanofibers in various medical and paramedical applications. CONCLUSIONS

This contribution shows the advantage of using three-way electrospinning when preparing mats from silk solution containing HAp and silver nitrate, which are able to embed perfectly in fibers. It is shown for the first time, that three-way electrospinning techniques enable obviating the ionic interaction between the HAp and silk solution, without being reacting with each other and hence no coagulation can occur. The silver NPs can be used to facilitate the antimicro-

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bial properties to nanofibers. The cell toxicity results after culturing the fibroblasts in presence of nanofibers indicate nontoxicity behavior with 0.5% used silver nitrate and nanofibers with 1.0 and 1.5% possess the toxic behavior toward NIH 3T3 fibroblasts. Furthermore, future studies are needed to figure out the potency of these nanofibers for bone tissue regeneration. REFERENCES 1. Silver S. Bacterial silver resistance: Molecular biology and uses and misuses of silver compounds. FEMS Microbiol Rev 2003;27: 341–353. 2. Abou El-Nour KMM, Eftaiha A, Al-Warthan A, Ammar RAA. Synthesis and applications of silver nanoparticles. Arabian J Chem 2010;3:135–140. 3. Rai MK, Deshmukh SD, Ingle AP, Gade AK. Silver nanoparticles: The powerful nanoweapon against multidrug-resistant bacteria. J Appl Microbiol 2012;112:841–852. 4. Tian J, Wong KKY, Ho CM, Lok CN, Yu WY, Che CM, Chiu JF, Tam PKH. Topical silver nanoparticles reduce systemic inflammation and promote wound healing. Chem Med Chem 2007;2:129–136. 5. Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci 2007; 32:991–1007. 6. Mandal BB, Das S, Choudhury K, Kundu SC. Implication of silk film RGD availability and surface roughness on cytoskeletal

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