Electrospun soy protein nanofiber scaffolds for tissue regeneration.

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Electrospun soy protein nanofiber scaffolds for tissue regeneration Karpagavalli Ramji and Ramille N Shah J Biomater Appl published online 7 April 2014 DOI: 10.1177/0885328214530765 The online version of this article can be found at: http://jba.sagepub.com/content/early/2014/04/07/0885328214530765 A more recent version of this article was published on - Aug 18, 2014

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Article

Electrospun soy protein nanofiber scaffolds for tissue regeneration

Journal of Biomaterials Applications 0(0) 1–12 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214530765 jba.sagepub.com

Karpagavalli Ramji1 and Ramille N Shah1,2

Abstract Electrospun fibers with an average fiber diameter in the nanometer range were prepared from soy protein isolate to develop scaffolds for tissue engineering applications. Poly(ethylene oxide) was added to facilitate fiber formation. The influence of processing parameters such as applied voltage, soy protein isolate and poly(ethylene oxide) concentrations, and poly(ethylene oxide) molecular weight on electrospun fiber morphology was investigated. Resulting soy protein isolate/poly(ethylene oxide) mats were carbodiimide crosslinked to increase construct robustness. Mechanical properties and in vitro biocompatibility of crosslinked electrospun scaffolds were evaluated. Soy protein isolate/poly(ethylene oxide) fiber diameters ranged between 50 and 270 nm depending on both electrospinning and solution parameters. The Young’s modulus for 7% soy protein isolate/3% poly(ethylene oxide) and 12% soy protein isolate/3% poly(ethylene oxide) electrospun scaffolds were 75 and 252 kPa, respectively. Human mesenchymal stem cell studies showed successful cell adhesion and proliferation on the soy protein isolate/poly(ethylene oxide) fibers. The structural and biological properties of these soy protein isolate electrospun scaffolds suggest their potential applications in tissue engineering. Keywords Soy protein isolate, electrospinning, nanofibers, scaffolds, tissue regeneration

Introduction Substantial efforts have been invested to develop biodegradable polymer scaffolds suitable for tissue engineering applications, especially those that can mimic the nanostructural architecture found in the natural extracellular matrix.1,2 Electrospinning is a versatile technique to produce nonwoven fiber mats with porous structure and excellent pore interconnectivity from natural and synthetic polymer solutions with fiber diameters ranging from 2 nm to several micrometers using electrical forces. A variety of natural and synthetic biodegradable polymers, including collagen, dextran, silk, poly(lactic acid) (PLA), poly(D,L-lactide-co-glycolide) (PLGA), and poly("-caprolactone) (PCL), have been successfully electrospun to form nanofibrous scaffolds for different tissue engineering applications.3–5 In the present study, we focus on the electrospinning of soy protein isolate (SPI) to fabricate tissue engineering scaffolds. Soy protein consists of amino acids of aspartic acid (aspargine) and glutamic acid (glutamine), nonpolar amino acids (glycine, alanine, valine, and leucine), basic amino acids (lysine and arginine), and less

than 1% of cysteine.6 The presence of high amounts of reactive groups, such as –NH2, –OH, and –SH, makes soy protein suitable for chemical, physical, and enzymatic modifications toward the diverse requirements of specific biomedical applications.7 Soy has additional advantages such as being of nonanimal origin (i.e. plant derived), it is an abundant and natural renewable resource, and it has a greater storage stability compared to other biodegradable polymers and natural proteins available for biomedical applications. Vaz et al. developed soy-based membranes, microparticles, and thermoplastics for the production of drug delivery carriers to be used in wound dressings, barrier membranes, and for oral, nasal, and subcutaneous drug delivery.8

1

Department of Materials Science and Engineering, Northwestern University, Evanston, United States 2 Institute for BioNanotechnology in Medicine and Department of Surgery, Northwestern University, Chicago, United States Corresponding author: Karpagavalli Ramji, Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208, United States. Email: [email protected]



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Soy-based bone filler has been obtained by thermosetting of defatted soybean curd and has been shown to reduce the chronic inflammatory response caused by protracted activation of macrophages and promote bone regeneration by stimulating bone cells.9 Vega Lugo and Lim investigated the electrospinnability of SPI with the addition of various polymers such as poly(ethylene oxide) (PEO) (Mw 900,000), PLA, and surfactants like triton X-100.10,11 The authors evaluated the electrospinnability of soy protein/PEO/ PLA blend with the addition of allyl isothiocyanate for controlled drug release. Cho et al. fabricated SPI/polyvinyl alcohol (PVA) electrospun fibers and analyzed their mechanical properties and biodegradability.12,13 They reported that the mechanical strength of electrospun nanofiber mats decreased gradually as the SPI content increased. Phiriyawirut et al. successfully obtained electrospun fibers from zein/SPI (95%/ 5%) blend.14 They concluded that smaller diameter fibers can be produced by increasing either electrostatic distance or voltage. Lin et al. obtained stable electrospun scaffolds with SPI/PEO (Mw 106) dissolved in 1,1,1,3,3,3,-hexafluoro-2-propanol and evaluated the cytocompatibility of these scaffolds.15 They reported that these soy-derived scaffolds supported the adhesion and proliferation of cultured primary human dermal fibroblasts. Xu et al. investigated the properties of SPI:PEO electrospun membranes.16 The authors found that SPI and PEO are miscible within the nanofibers, and that the nanofibers were superhydrophilic, which could be a desirable property for many applications. The goal of this work was to optimize electrospun fiber morphologies of SPI and PEO composites (dissolved in aqueous solvents) for the successful attachment and growth of human mesenchymal stem cells (hMSC) for tissue engineering applications. One of the aims was to better understand the effect of SPI concentration, PEO concentration and molecular weight, and postcrosslinking on fiber morphology and overall mechanical properties of the resulting electrospun scaffolds. We therefore investigated the influence of PEO on the electrospinnability of SPI and explored the effect of solution formulation on viscosity, surface tension, and SPI electrospun fiber morphology. Fourier transform infrared spectroscopy (FTIR) measurements were carried out to analyze the integrity of SPI in the uncrosslinked and crosslinked electrospun scaffolds. Furthermore, successful crosslinking of the electrospun scaffolds was verified using FTIR peak analysis. A detailed microstructural, mechanical, and biocompatibility assessment of the resulting SPI electrospun scaffolds was carried out to determine its potential as a matrix material for tissue regeneration.

Materials and methods Materials Commercial SPI (protein content >90%) from Now Foods, IL, USA was used for the electrospinning experiments. PEO with an average molecular weight of 1 105, 1 106, and 8 106 Da; sodium hydroxide (NaOH); 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC); N-hydroxysulfosuccinimide (NHS); glutaraldehyde; and sucrose were purchased from Sigma-Aldrich, USA.

Electrospinning Solutions containing various concentrations of SPI and PEO were prepared by heat treating 60 C at alkaline conditions (1% NaOH). For example, to prepare 7% SPI/3% PEO solution, 7 wt.% SPI, 3 wt.% PEO, 1 wt.% NaOH, and 89 wt.% water were mixed and heat treated at 60 C for an hour. SPI/PEO solution was placed in a syringe and mounted on a syringe pump (Model 100, KD Scientific Inc., USA). A 20gauge blunt end needle was used for electrospinning. Applied voltage was varied from 15 to 27 kV (Figure 1) to obtain smooth fibers (Model ES30, Gamma High Voltage Research Inc., Florida, USA). Upon applying positive voltage, a charged jet of SPI/PEO solution was ejected from the tip of the needle and deposited on a piece of aluminum foil affixed to the grounded collector to form a nonwoven fiber mat. The solution flow rate and working distance between needle tip and collector were optimized to 0.004 ml/min and 18 cm, respectively.

Surface tension and viscosity measurements Surface tension of SPI/PEO solutions were measured using drop shape analysis system (DSA100, KRUSS Advanced Surface Science, Germany) with a 14-gauge blunt tip needle. Viscosity of SPI/PEO solutions was determined using Anton Paar Physica MCR 300 modular compact rheometer. Viscosity was measured at shear rates of 10 through 1000 s1 at 25 C. A coneplate with 50 mm diameter and 2 angle was used for viscosity measurements (CP 50-2).

Scanning Electron Microscopy (SEM) and fiber diameter measurements SPI/PEO electrospun fiber morphology and hMSC attachment and spreading on electrospun scaffolds were observed using SEM (Hitachi S-4800-II) at an accelerating voltage of 3 kV after 9 nm thick osmium sputter coating. SEM images were taken from two different samples for each condition and 25 measurements of fiber diameter were carried out randomly at various



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places of each sample using an image analysis software INCA to calculate the average fiber diameter. For the average fiber diameter measurements, areas with beads and spindles were not included.

FTIR measurements FTIR measurements were carried out for SPI powder, PEO (1 MDa) powder, uncrosslinked and crosslinked SPI/PEO electrospun scaffolds to determine any changes in soy protein structure, degree of crosslinking, and presence of PEO before and after the crosslinking treatment. FTIR spectra were recorded in the midinfrared range (4000–400 cm1) using attenuated total reflectance Fourier transform infrared spectrometry (Thermo Nicolet Nexus 870). Typically, 32 scans were signal-averaged for a single spectrum at a resolution of 8 cm1. The spectra were analyzed using Omnic software to obtain quantitative peak information.

Tensile tests Electrospinning was carried out for 1.3 h to produce electrospun mats. Dog-bone shaped specimens of 47 3 t mm (length width thickness) were cut from SPI/PEO electrospun mats and were crosslinked using 10 mM EDC/4 mM NHS. After crosslinking electrospun scaffolds were hydrated for 1 h before tensile testing. Tensile tests were performed at room temperature with LFPlus mechanical tester integrated with NexygenPlus software (Lloyd Instruments Ltd, UK). Tests were performed using a cross-head speed of 3 mm/min. A small initial loading of 0.002 kN was applied on the strips to remove any waviness that might be present after fixing between the cross-head (i.e. to ensure sample is taut). This initial load did not cause any sensible extension beyond removing the above mentioned defects. The electrospun samples were stretched to failure.

hMSC culture hMSCs were purchased from Lonza Walkersville, Inc., MD (# PT-2501). Cells were cultured in mesenchymal stem cell growth medium base (Lonza Walkersville, MD) with 10% fetal bovine serum, 2 mM L-glutamine, and 100 IU/ml penicillin–streptomycin. Electrospinning of 7% SPI/3% PEO (1 MDa) and 12% SPI/3% PEO (1 MDa) solutions were carried out for 1.3 h and the electrospun mat was punched out into circular shape scaffolds with 6 mm diameter. The thickness of the uncrosslinked scaffolds obtained for 7% SPI/3% PEO and 12% SPI/3% PEO were 289 and 903 mm, respectively. After EDC/NHS crosslinking, these scaffolds were immersed in 100% ethanol to remove excess

crosslinking reagents. Cell culture studies were performed in duplicate. Before cell seeding, the scaffolds were washed twice in 70% ethanol and three times in 1 DPBS buffer (Dulbecco’s phosphate buffered saline) (Hyclone) each for 10 min. The scaffolds were sterilized under ultraviolet radiation for 30 min before cell seeding. Passage 5 hMSCs were seeded at a density of 50,000 cells/well. Cells were cultured in humidified 5% CO2 incubator kept at 37 C. Cell culture media were exchanged every 3–4 days. Cell viability and spreading were studied on 1 and 7 days of culture.

Cell viability, picoGreen dsDNA quantitation, and cell morphology analysis Cell viability was assessed using a live/dead assay kit (Invitrogen). Separate live and dead fluorescence images were taken using mercury lamp (CHIU Technical Corporation) Nikon Eclipse TE 2000-U microscope in conjunction with blue (450–490 nm) and green (510–560 nm) filters. The color images were then merged using Metaview imaging software. Total dsDNA content in each scaffold was determined using Quant-iTTM PicoGreen dsDNA assay kit (Invitrogen). To digest, the scaffolds were incubated overnight at 37 C in 100 mg/ml Proteinase K/Tris–HCl buffer solution. dsDNA quantitation was carried out according to the manufacturer’s protocol using a Spectramax M5 microplate reader (Molecular Devices Corporation, CA). Cell attachment and spreading on SPI electrospun scaffolds was investigated using SEM. To perform SEM imaging, the scaffolds were fixed in 2% glutaraldehyde and 3% sucrose for 2 h.17 After fixing, scaffolds were dehydrated in sequential ethanol series (70, 80, 90, and 100%), then critically point dried (Tousimis Research Corporation, MD), and sputter coated with osmium (9 nm).

Results and discussion Change in electrospun morphology with SPI and PEO concentrations Table 1 presents the electrospinnability of SPI and PEO alone, as well as various SPI/PEO solutions investigated and their electrospun morphology. Even though the mixture of SPI and PEO successfully formed fibers, neither SPI nor PEO alone at the same concentrations was able to form electrospun fibers. This result indicates that the molecular interactions between SPI and PEO significantly influence the electrospinning properties. Prior to electrospinning, SPI/PEO solutions were prepared by heat treating them at 60 C in alkaline conditions (pH 13) for 2 h. Under these conditions SPI is more unfolded and hydrolyzed18 and therefore PEO, which has both



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Journal of Biomaterials Applications 0(0) Table 1. Electrospinnability of SPI/PEO solutions (electrospinning conditions: applied voltage 27 kV, working distance 18 cm, and flow rate 0.004 ml/min). Solution chemical composition

Electrospun fiber morphology

7% SPI 5% 100 kDa PEO 10% 100 kDa PEO 15% 100 kDa PEO 20% 100 kDa PEO 3% 1 MDa PEO 5% SPI/5% 100 kDa PEO 7% SPI/5% 100 kDa PEO 10% SPI/5% 100 kDa PEO 12% SPI/5% 100 kDa PEO 7% SPI/10% 100 kDa PEO 7% SPI/15% 100 kDa PEO 7% SPI/20% 100 kDa PEO 7% SPI/3% 100 kDa PEO 7% SPI/3% 1 MDa PEO 7% SPI/3% 8 MDa PEO

Droplets Droplets with few short fibers Short fibers with droplets Fibers with many droplets, beads, and spindles Continuous fibers; fibers merged at the joints Fibers with many spindles and droplets; fibers merged at the joints Short fibers with droplets Continuous fibers with beads and spindles Continuous fibers with lesser beads and spindles Continuous fibers Fibers with lesser beads and spindles Continuous fibers with lesser beads and spindles Continuous fibers Short fibers with droplets Continuous fibers Continuous fibers

(a)

(b)

1 µm

1 µm (d)

80

Average fiber diameter (nm)

(c) 60 40 20 0

1 µm

10

20 Applied voltage (kV)

30

Figure 1. Influence of applied voltage on electrospun fiber morphology of 7% SPI/5% 100 kDa PEO (a) 15 kV, (b) 20 kV, (c) 27 kV, and (d) variation in average fiber diameter.

hydrophilic ether oxygen and hydrophobic methylene segments, can interact with the amino acids of SPI protein through ionic, hydrogen, and hydrophobic interactions. PEO has electron-rich oxygen in the backbone polymer chain that can ionically interact with positively charged lysine, arginine, and histidine residues of soy protein. Hasek reported such an interaction between polyethylene glycol and proteins.19 Ji et al. also observed

ionic interactions between PEO and denatured SPI via oxygen atoms of PEO and positively charged amino groups of SPI.20 These interactions between SPI and PEO may explain the ability to electrospin mixtures of the two solutions and not the individual polymer solutions. Typical fiber structures exhibiting the effect of SPI and PEO concentrations are shown in Figures 2 and 3,



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Figure 2. Change in electrospun fiber morphology with SPI concentration: (a) 5% SPI/5% 100 kDa PEO, (b) 7% SPI/5% 100 kDa PEO, (c) 10% SPI/5% 100 kDa PEO, (d) 12% SPI/5% 100 kDa PEO, (e) variation in average fiber diameter, and (f) surface tension and viscosity.

respectively. The results revealed that electrospun fiber morphology was affected by both SPI and PEO concentrations. Beaded structures with short fibers were produced for solutions containing lower concentrations of either SPI or PEO. If the concentration of SPI or PEO was increased, the structural features changed from beaded fibers with round beads to beaded fibers with spindle-like beads to more uniform fibers with lesser defects. Increasing the concentration of SPI is beneficial for the formation of continuous uniform fibers due to unfolding of protein chains during heat treatment of the SPI/PEO solution at high pH (pH 13). The adjacent portions of proteins start to repel each other due to high charge density at extreme alkali pH that leads to irreversible unfolding of protein molecules to form a more fibrillar structure. These conditions make the soy protein remarkably stable against thermal aggregation. The refolding of protein molecules even at room temperature is inhibited at this high pH condition.21 The more the number of unfolded protein chains (i.e. higher concentrations of SPI), the easier the formation of continuous fibers during the electrospinning process. The fact that continuous fibers with lesser number of beads formed with increasing concentration of PEO (holding SPI concentration constant) indicates

that PEO enhanced polymer chain entanglement, which can prevent the polymer jet from breaking up into droplets.22 These results therefore reveal that the electrospinnability of SPI/PEO solutions is possible due to the synergistic effect of unfolded hydrolyzed SPI protein chains and entanglement of PEO polymer chains. The solution surface tension and viscosity are the critical factors that affect spinnability and morphology of electrospun fibers. The viscosity of 7% SPI, 5% PEO (100 kDa), and the mixture of 7% SPI/5% PEO (100 kDa) were measured as 0.004, 0.020 and 0.094 Pa.s, respectively. The fact that the viscosity of the SPI/PEO mixture was higher than either the 7% SPI or 5% PEO alone is another indication that there is entanglement of the protein and polymer chains.23 The entanglements act like nodes of covalently bonded network junctions that restrict the motion of the protein and polymer chains leading to the increased viscosity of the mixture. Figures 2 and 3 include the surface tension and viscosity for the SPI/PEO solutions studied. A slight decrease in surface tension and a significant increase in viscosity were observed while increasing the concentration of either SPI or PEO. Continuous fibers were formed with lesser defects for solutions that had lower surface tension and higher viscosity. In the case of lower



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Figure 3. Change in electrospun fiber morphology with PEO concentration: (a) 5% 100 kDa PEO/7% SPI, (b) 10% 100 kDa PEO/7% SPI, (c) 15% 100 kDa PEO/7% SPI, (d) 20% 100 kDa PEO/7% SPI, (e) variation in average fiber diameter, and (f) surface tension and viscosity.

viscosity solutions, surface tension plays a dominant role and thus beads or beaded fibers are formed due to the tendency of solvent molecules to congregate to form droplets.6 At higher viscosity the interaction between protein and polymer chains is great enough to overcome the surface tension, resulting in continuous fiber formation. Analysis of electrospun fiber morphology obtained through SEM showed that the average fiber diameter of electrospun scaffolds increased from 30 to 90 nm with increasing SPI concentration (Figure 2(e)) and from 30 to 150 nm with increasing PEO concentration (Figure 3(e)). The increase in average fiber diameter with increasing SPI or PEO concentration is due to greater entanglement of macromolecules in the mixed solution, which increases the solution viscosity that causes the formation of larger fiber diameters. In all SPI and PEO mixture formulations a second population of smaller size fibers with diameters ranging from 15 to 25 nm was observed. These smaller diameter fibers may be formed due to the splaying of polymer jet during the electrospinning process.24

Influence of PEO molecular weight The average fiber diameter and inter-fiber spacing of SPI/PEO electrospun fibers increased significantly with increasing PEO molecular weight (Figure 4). During electrospinning, the charged jet ejected from the Taylor cone is subjected to tensile stresses and undergoes significant elongational flow. The nature of this elongational flow determines the degree of subsequent splitting and splaying of the jet. This elongational flow is dependent on the elasticity of the solution, which depends on the polymer molecular weight. It has been reported that the relaxation of the polymeric chain becomes more difficult as the molecular weight increases and the jet splitting and splaying processes are not as effective.25 As a result, the fiber diameter increases with increasing PEO molecular weight. Furthermore, rheology results showed an increase in solution viscosity and decrease in surface tension with increasing PEO molecular weight (Figure 4(f)) leading to continuous fibers with lesser defects and larger fiber diameters. Since it was found that using higher



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Figure 4. Influence of PEO molecular weight on electrospun fiber morphology: (a) 3% 100 kDa PEO/7% SPI, (b) 3% 1 MDa PEO/7% SPI, (c) 3% 8 MDa PEO/7% SPI, (d) higher magnification of 3% 1 MDa PEO/7% SPI electrospun fiber which is used for tensile tests and cell culture studies, (e) variation in average fiber diameter, and (f) surface tension and viscosity.

molecular weight PEO required less amount of PEO to form continuous and uniform fibers with SPI, the lower concentration of higher molecular weight PEO (i.e. 3% 1 MDa PEO) was used for cell culture studies.

Effect of EDC/NHS crosslinking Prior research using EDC and NHS as crosslinking agents for collagen scaffolds has demonstrated that EDC/NHS crosslinked material is noncytotoxic in vitro and biocompatible in vivo.26,27 EDC/NHS was therefore used to crosslink SPI/PEO electrospun scaffolds to enhance structural integrity and prevent dissolution of the scaffold upon hydration. Crosslinking of SPI using EDC/NHS involves the activation of carboxylic acid groups present in the polypeptide chain followed by reaction with free amine groups of another polypeptide chain.28 Vaz et al. determined the carboxylic group content for soy protein to be 1.08 mmol/g protein.29 In the present study, EDC/ NHS molar ratio was calculated based on this reported carboxylic acid group content for soy protein. EDC and NHS concentrations and duration of crosslinking

were varied and optimized (data not shown) to obtain scaffolds that did not dissolve when placed in media. Electrospun scaffolds with stable structures were obtained when crosslinked at a molar ratio of 10:4:1 EDC to NHS to COOH for 24 h. The electrospun scaffolds were immersed in ethanol for 12 h after crosslinking to remove excess reagents. The fiber morphology of SPI electrospun scaffolds treated with EDC/NHS and only ethanol is shown in Figure 5. For the ethanol-only treated samples, SEM micrographs revealed significant swelling of fibers (3 the original diameter) as well as coalescing of the fibers. In contrast, fibers were still distinct with significantly less swelling (1.5 the original diameter) and bundling for EDC/NHS crosslinked scaffolds. The fibers of the uncrosslinked electrospun scaffolds (Figure 4(b)) stay separated due to electrostatic repulsion, whereas the crosslinking treatment resulted in bundling of some of the fibers with significant increase in average fiber diameter (Figure 5(c)). Liu et al. reported similar observations of increased electrospun fiber diameter for silk-fibroin fiber mats after EDC crosslinking.30 They concluded that the amide bonds formed during



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(d) 100 Porosity (%)


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60 40 20 0

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7% SPI/3%PEO 12% SPI/3%PEO

Figure 5. Electrospun fiber morphology of (a) ethanol immersed 7% SPI/3% 1 MDa PEO, (b) EDC/NHS crosslinked 7% SPI/3% 1 MDa PEO, (c) change in average fiber diameter, and (d) porosity before and after EDC/NHS crosslinking.

EDC crosslinking inhibit the retraction of the nanofibers after crosslinking that leads to increased fiber diameter. This phenomenon may also be occurring in this SPI electrospun fiber system along with fiber bundling. The porosity of SPI electrospun scaffolds before and after crosslinking was measured using image analysis as described in ASTM E2109–01. Porosity was determined by superimposing a grid on SEM images using ImageJ software and counting the number of grid line intersections that fall within a pore.31 This number was subsequently divided by the total number of line intersections in the grid to obtain the percent porosity. Results revealed that there was no significant decrease in porosity after crosslinking (Figure 5(d)). This image analysis method was chosen to measure the porosity as opposed to an intrusion method like mercury porosimetry due to fragility of the electrospun scaffolds.32

FTIR measurements FTIR analysis was used to determine any changes in soy protein structure from the electrospinning process, the presence of PEO before and after the crosslinking treatment, and to confirm successful crosslinking of the electrospun scaffolds. Figure 6(a) shows the resulting FTIR spectra for SPI powder, PEO powder, and uncrosslinked and crosslinked electrospun scaffolds of 7% SPI/3% PEO and 12% SPI/3% PEO. An increased spectral intensity was observed for uncrosslinked SPI/ PEO electrospun samples as compared to the individual SPI and PEO powder components. SPI powder, uncrosslinked SPI electrospun, and crosslinked SPI electrospun samples all showed peaks at 3300 cm1 (amino acid peak) and 2850 cm1 (–CH2 stretching).33

A detailed view of the amide bands for SPI powder, uncrosslinked and crosslinked electrospun 7% SPI/ 3% PEO (Figure 6(b)) showed that all samples had amide I and II bands in the same ranges (e.g. between 1600–1700 cm1 and 1480–1575 cm1). The fact that all SPI samples have similar spectral bands indicates that neither electrospinning nor crosslinking affected the original SPI protein structure. Interestingly, the SPI powder and crosslinked electrospun samples, but not the uncrosslinked electrospun samples, showed an amide III peak centered at 1222 cm1 indicating the presence of b-sheets.33 This suggests that conditions needed to make the SPI slurry for electrospinning (i.e. basic conditions and heat treatment) may have decreased the b-sheet structure of the original soy protein, and that crosslinking of the electrospun scaffolds may have recovered some of the lost b-sheet structure. Comparison of the PEO spectra to those of the electrospun samples shows the presence of peaks corresponding to –CH2 twisting vibration (1238 and 1276 cm1) for PEO34 and uncrosslinked electrospun samples, but not for crosslinked electrospun samples. This observation confirms the presence of PEO in the uncrosslinked electrospun samples, but indicates that the PEO may have leached out during the crosslinking process. The leaching of high molecular weight PEO (8 MDa) from electrospun collagen scaffolds during EDC crosslinking in ethanol has also been previously observed by Buttafoco et al.35 Finally, to verify successful crosslinking of the SPI electrospun scaffolds with EDC/NHS, the absorption peak area ratio of 3300 cm1, which is assigned to N–H stretching vibrations for alkyl and aryl amines, to amide I stretching at 1650 cm1 was determined for



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7%SPI/3%PEO electrospun

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8 7 6 5 4 3 2 1

7%SPI/3%PEO electrospun sample 12%SPI/3%PEO electrospun sample 7%SPI/3%PEO crosslinked sample 12%SPI/3%PEO crosslinked sample

0

Figure 6. (a) FTIR spectra for SPI powder, PEO powder, uncrosslinked and crosslinked electrospun 7% SPI/3% PEO and 12% SPI/3% PEO, (b) peaks in the range of 1180–1750 cm1 for SPI powder, PEO powder, uncrosslinked and crosslinked electrospun 7% SPI/3% PEO, and (c) 3300 cm1: 1650 cm1 peak area ratio.

Table 2. Tensile properties of crosslinked SPI/PEO electrospun scaffolds at hydrated condition, n ¼ 3. Electrospun

Young’s modulus (kPa)

Tensile strength (MPa)

Strain at break (%)

7% SPI/3% PEO 12% SPI/3% PEO

110 6** 171 21**

0.06 0.01**** 0.17 0.006****

60 3*** 70 12***

** ¼ p-value < 0.01; *** ¼ p-value < 0.001; **** ¼ p-value < 0.0001.

the uncrosslinked and crosslinked SPI electrospun scaffolds (Figure 6(c)). A decrease in the 3300–1650 cm1 peak ratio indicates a decrease in –NH2 groups and an increase in amide linkages.36 Figure 6(c) shows a decreased ratio for the crosslinked electrospun scaffolds compared to the corresponding uncrosslinked scaffolds indicating effective crosslinking (between –COOH and –NH2 groups). Results revealed a slightly higher degree

of crosslinking for 7% SPI/3% PEO (79%) compared to 12% SPI/3% PEO (70%).

Tensile properties of crosslinked SPI electrospun scaffolds Table 2 presents the tensile properties of hydrated crosslinked 7% SPI/3% PEO and 12% SPI/3% PEO



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electrospun scaffolds (spun for 1.3 h). A representative tensile strain–stress curve for 12% SPI/3% PEO is shown in Figure 7. Young’s modulus (E0 ) was found to be 110 6 and 171 21 kPa for 7% SPI/3% PEO and 12% SPI/3% PEO, respectively. The tensile strength for 7% SPI/3% PEO scaffold was 0.06 0.01 MPa and for 12% SPI/3% PEO scaffold was 0.17 0.006 MPa. The higher modulus and tensile strength of the 12% SPI condition may be due to the higher protein content as well as the greater average fiber diameter compared to the 7% SPI condition

(Figure 2). Interestingly, the strain at break was similar for both 7% SPI/3% PEO and 12% SPI/3% PEO scaffolds (Table 2). In contrast to Cho et al. tensile strength increased with increasing SPI concentration.13 They reported a decrease in SPI/PVA electrospun fiber diameter with increasing SPI concentration that resulted in decreased mechanical strength. In our studies the tensile strength of SPI/PEO electrospun scaffolds increased with increasing SPI concentration, which is attributed to the increase in electrospun fiber diameter.

15 Day 1 Concentration, ng/ml

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Figure 7. Representative tensile strain–stress curve: 12% SPI/ 3% PEO.

(a) i

Figure 9. PicoGreen dsDNA quantitation (n ¼ 4;* ¼ p-value < 0.05; ** ¼ p-value < 0.000001) of hMSCs seeded on 7%SPI/3% PEO and 12%SPI/3% PEO after 1 and 7 days of culture.

(a) ii

100 µm

100 µm (b) ii

(b) i

4 µm

4 µm

Figure 8. (a) Fluorescence micrographs of live/dead stained hMSCs and (b) SEM micrographs of hMSCs attached and spread on 12%SPI/3% PEO electrospun after (i) 1 and (ii) 7 days of culture.



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hMSC culture

SEM characterization revealed cell spreading and interaction with the electrospun nanofibers. The results of the present study suggest that SPI/PEO electrospun scaffolds may present a new type of protein-based nanofiber scaffold for tissue engineering applications.

Cell culture studies using hMSCs were carried out for crosslinked 7% SPI/3% PEO and 12% SPI/3% PEO electrospun scaffolds to evaluate their ability to support cell viability and growth. Figure 8 shows hMSC viability (fluorescence image) and cell morphology (SEM micrograph) on 12% SPI/3% PEO electrospun scaffold after 1 and 7 days of culture. Cells were spread on the surface of nanofiber mat exhibiting their normal phenotypic shape. SEM images verify successful hMSC attachment and spreading on SPI electrospun scaffolds. Clear interactions of cytoplasmic extensions of the hMSCs can be seen with the SPI fibers at both time points. Cells appeared to be completely integrated into the structure of the scaffold on day 7. Figure 9 shows the quantitative DNA amounts for electrospun samples. DNA amounts on day 1 indicate that the 12% SPI scaffolds resulted in a significantly higher cell number compared to 7% SPI scaffolds (threefold higher). This may be due to higher SPI content providing more protein-cell binding sites37 as well as the greater thickness of the 12% SPI scaffolds. The thickness of the 12% SPI scaffolds was about threefold thicker (903 mm) compared to the 7% SPI scaffolds (289 mm). DNA quantification on day 7 indicated higher cell proliferation on 7% SPI scaffolds (2 day 1 amount), while little proliferation occurred on the 12% SPI scaffolds (1 day 1 amount). The less proliferation observed for the 12% SPI scaffolds may be because cells in the observed clusters were already locally crowded, even though there was space surrounding the clusters, which may have affected their ability to spread out to other areas, hindering their ability to proliferate. The collective results of this study demonstrate that novel SPI electrospun scaffolds can support cell viability and proliferation and may have potential use in regenerative medicine applications.

Conclusions SPI/PEO solutions have been successfully electrospun into fibers in the range of 50–270 nm. The electrospun fiber diameter was found to increase with increasing concentration of either SPI or PEO, as well as with increasing PEO molecular weight. More robust SPI/ PEO electrospun scaffolds were successfully obtained through carbodiimide crosslinking. Even though the fiber diameter increased after crosslinking, no significant change in porosity was observed. Young’s modulus was higher for crosslinked electrospun scaffolds and those containing higher SPI content. Cell culture studies showed that the SPI/PEO nanofiber scaffolds could support hMSC attachment, viability, and proliferation.

Acknowledgements Biological and chemical analysis was performed in the Equipment Core Facility of the Institute for BioNanotechnology in Medicine at Northwestern University. SEM experiments were performed in the EPIC facility of NUANCE center at Northwestern University. The authors would like to thank Prof. Kenneth R. Shull and Prof. Wesley R. Burghardt for providing surface tension and viscosity experimental facilities.

Conflict of interest None declared.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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