Biohybrid nanofiber constructs with anisotropic biomechanical properties

Biohybrid nanofiber constructs with anisotropic biomechanicalproperties

W. Bonani,1,2 D. Maniglio,1 A. Motta,1 W. Tan,2,3 C. Migliaresi1

1Department of Materials Engineering and Industrial Technologies, and BIOtech Research Center, University of Trento,

Via Mesiano 77, 38100 Trento, Italy2Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado3Department of Pediatrics-Cardiology, Center for Bioengineering, University of Colorado at Denver, Aurora, Colorado

Received 15 December 2009; revised 13 June 2010; accepted 3 September 2010

Published online 10 December 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31763

Abstract: Synthetic implant materials often lack of the

anisotropic mechanical properties and cell-interactive surface

which are shown by natural tissues. For example, engineered

vascular grafts need to be developed to address the mechani-

cal and biological problems associated with the graft materi-

als. This study has demonstrated a double-electrospinning

fabrication process to produce a poly(e-caprolactone)-fibroinmultilayer composite which shows well-integrated nanofi-

brous structure, endothelial-conducive surface and aniso-

tropic mechanical property, suitable as engineered vascular

constructs. Electrospinning parameters such as voltage,

solution concentration, feed rate, and relative humidity were

optimized to obtain defect-free, uniform nanofibers. To mimic

the different mechanical properties of natural vessels in the

circumferential and longitudinal directions, a rotating cylinder

was used as collector, resulting in the production of

constructs with anisotropic properties. The combination of

the collector shape and the collector rotation allows us to

produce a tubular structure with tunable anisotropic mechan-

ical properties. Fourier transform infrared spectroscopy,

differential scanning calorimetry, and uniaxial tensile tests

were used to characterize the electrospun constructs. Cell

cultures with primary endothelial cells demonstrated that

cells showed spread morphology and strong adhesion on

fibroin richer surfaces. The platform for producing robust

multilayer scaffolds with intermixing nanofiber structure, tun-

able anisotropy ratio, and surface with specific compositions

may hold great potential in tissue engineering applications.VC 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl

Biomater 96B: 276–286, 2011.

Key Words: nanofibers, fibroin, anisotropy, electrospinning

INTRODUCTION

The proper design of a scaffold that supports tissueregeneration has to take in account for a variety of factorssuch as mechanical properties, surface properties, mor-phology, pore size and distribution, biocompatibility, andbiodegradability,1,2 Mechanical properties on tissue rege-neration and cell growth are particularly important intissues such as bone, muscles, cartilage, blood vessels, andtendons.3

Vascular grafts are currently used to replace damaged oroccluded blood vessels for treatment of a number of vasculardiseases such as coronary vascular diseases. Many patientsthat require vascular grafting do not have suitable autolo-gous grafts and thus require synthetic grafts. Syntheticvascular grafting materials currently in clinical use are oftenmade from stiff, bioinert materials such as expanded polytet-rafluoroethylene (ePTFE) and dacron. When these syntheticgrafts are used to replace small-diameter (<6 mm) vessels,low patency or high failure rates occur.4 Grafting pathologyis often characterized by anastomotic intimal hyperplasiaand thrombosis, which are induced by improper hemody-namic, inflammatory or coagulatory conditions around

grafts. A leading cause of these adverse conditions is themechanical and biological properties of the syntheticgraft materials.5

Vascular tissue engineering offers a promising alterna-tive approach to address the needs for small-diametervascular grafts by overcoming the mechanical and/orbiological problems associated with the current materials.6

To avoid adverse hemodynamic conditions, mechanicalproperties of tissue engineering grafts must closely matchthose of native arteries, meanwhile the grafts should besufficiently strong. Many studies have demonstrated theanisotropic mechanical property of natural arteries,7,8 higherstiffness in the longitudinal direction can avoid an excessivestimulation of anastomotic regions, while being compliant inthe circumferential direction to match the compliance ofnative artery can avoid flow disturbance.9,10

Therefore, one of the goals in this study is to create astructure that reproduces anisotropic mechanical propertiesof natural arteries. In addition, to provide anticoagulatoryand anti-inflammatory environments, the graft surface mustbe inherently antithrombogenic or conducive to endothelialcells. Furthermore, the presence of nondegradable grafts

Correspondence to: C. Migliaresi; e-mail: [email protected] grant sponsors: Innovative Grant Program of the University of Colorado at Boulder, CO and BDGE

276 VC 2010 WILEY PERIODICALS, INC.

always imposes long-term concerns about the syntheticmaterials. Therefore, an ideal tissue engineering graftscaffold should not only closely match the mechanical prop-erties of native arteries and be conductive to endothelialcells, but also be degradable as it provides a temporaryscaffold to support the growing tissue substitute until cellsproduce their own extracellular matrix. It has been shownthat a single material is difficult to simultaneously satisfy allthese requirements.

Electrospinning is an attractive technique for fabricatingtissue engineering scaffolds, due to the simplicity of the pro-cess and the capability of generating nanofibrous structuresthat mimic the natural extracellular matrix,11–13 A variety ofpolymers have been electrospun for biomedical applicationsproposed in a wide range of areas including skin, nerve,tendon, cartilage, bone, and blood vessel,14–16 Regeneratedsilk fibroin, one of the most promising biological polymers,is recently experimented in the field of tissue engineeringbecause of its favorable interactions with cells andproteins,17–19 Zhang et al. suggested that electrospun fibroinscaffolds held potential for explorations of tissue-engineeredvascular grafts because they sustained vascular cell viability,maintained cell phenotype and promoted cell reorgan-ization.20 In a recent study, Bondar et al.21 have shownthat endothelial cells recognize fibroin nanofibers throughintegrin-dependent mechanisms and, and the nanometricsilk fibroin scaffolds did not interfere with the formationof a differentiated and interconnected endothelial layer.Poly(e-caprolactone) (PCL) is a semicrystalline, bioresorb-able, aliphatic synthetic polyester. A number of implantablemedical devices are made of robust PCL. Electrospun PCLmaterials have been widely demonstrated for biomedicalapplications.22–24

In this study, we have demonstrated a novel multilay-ered biohybrid nanofiber scaffold made of silk fibroin andPCL. The scaffold is characterized by anisotropic mechanicalproperties, surface conducive to endothelial cells, anddegradation capacity. Anisotropic mechanical properties andsurfaces with different functionalities are achieved bysequentially and simultaneously electrospinning fibroin andPCL on a rotational mandrel, which forms a robust multilay-ered structure with nonhomogeneous compositions and aninterpenetrating fibrous layer in the transition region.

MATERIALS AND METHODS

MaterialsCocoons of Bombyx mori silkworm PN06 (Poli-ibridoNazionale 2006) were kindly supplied by Centro Sperimen-tale di Gelsibachicoltura (Como, Italy). Poly-e-caprolactone(PCL) with nominal average MW of 80,000 Da and poly(eth-ylene oxide) (PEO) with nominal average MW of 500,000Da were purchased from Sigma Aldrich S.r.l (Milan, Italy).Formic acid (98–100% purity) was also obtained fromSigma-Aldrich. N,N-dimethylformamide (DMF) minimum pu-rity 99.9%, and methylene chloride (DCM) minimum purityof 99.5% were purchased from BDH Italia (Milan, Italy). Allother substances were of analytical or pharmaceutical gradeand obtained from Sigma-Aldrich.

Preparation of regenerated Bombyx mori silk fibroinBombyx mori silk-fibroin water solutions were prepared aspreviously described.25 Briefly, to remove sericin, cocoonswere degummed by treating silk twice with 1.1 g/L and 0.4g/L Na2CO3 (anhydrous, minimum 99%) water solution at98!C for 1 h each time. This resulted in 1 L of solutioncontaining 10 g of silk fibroin, which were then washed indeionized water and air-dried. The extracted silk fibroinwas then dissolved in 9.3M LiBr (1 g fibroin in 10 mL LiBrsolution) at 65!C for 8–10 h. After that, the solution wasdialyzed in a Slide-A-Lyzer Cassette of 3500 MW (Pierce,Rockford, IL) against distilled water for three days in orderto remove the salt, and then filtered through a ceramic filterfoam (porosity <5 lm). Finally, water-soluble powders offibroin were prepared with a freeze-drying process.

ElectrospinningPreparation of spinning solutions. A series of fibroin solu-tions were prepared by dissolving regenerated silk-fibroinpowders in formic acid at a concentration of 8–16%(wt %). PCL was dissolved at a concentration of 9% (wt %)in a mixture of DCM/DMF with 3-to-1 volumetric pro-portion. PEO was dissolved at a concentration of 5% (wt %)in water.

Electrospinning of single-polymer materials. In this study,single-polymer materials and composite materials were pro-duced in flat sheet form or in tubular shape. The single-polymer nanofibrous materials were fabricated by a singleelectrospinning apparatus. A home-made electrospinning ap-paratus was composed of a Gamma High Voltage ResearchES30P-10W power supply (Gamma High Voltage ResearchInc., Ormond Beach, FL), a single syringe pump (Pump 11Plus – Harvard Apparatus, Crisel Instrument, Rome, Italy),and a flat aluminium collector to produce flat sheets or arotating cylindrical collector to produce tubular-shapedmaterials. A cylindrical collector with 20 mm in diameterwas rotated by a brushless rotating electric motor(BLF230C-A Oriental Motor Italia S.r.l., Milan, Italy). Rotationspeeds in the range of 50–2000 rpm were achieved. In addi-tion another collector with 4.96 mm in diameter rotating at100 rpm was used to produce constructs with a smaller di-ameter. To facilitate the detachment of the scaffold materials(fibroin or PCL), the collector was preventively coated witha thin veil of electrospun PEO. Fibroin and PCL fibers werecollected with needle-collector distances of 12 cm and 22cm, respectively. Syringes of 5 mL were filled with polymersolutions and fitted with a stainless-steel 18G blunt-endedneedle which is then connected to 25 cm flexible PTFEtubing.

Parametric study in the fibroin electrospinning process. Toproduce good-quality silk-fibroin nanofibers, several electro-spinning parameters were investigated and analyses werecarried out on evaluating their effects on fiber morphologyand diameter. The parameters of interest included appliedvoltage, solution concentration and feed rate, which variedin the range of 14–30 kV, 8–12% (wt %) and 1–12 lL/min,

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | FEB 2011 VOL 96B, ISSUE 2 277

respectively. In addition, the effect of environmental relativehumidity on fiber diameter was investigated. The electro-spinning processes were thus carried out in two differentenvironments with a relative humidity of 45–70%. Parame-ter configurations that resulted in defect-free fibers werecarried further for morphology, chemical and thermalcharacterizations.

Double-electrospinning system to produce nanofibrouscomposite scaffolds. Multilayered composite scaffoldswere produced by a new double-electrospinning system[Figure 1(a)] through sequentially and simultaneously electro-spinning fibroin and PCL on a rotational mandrel collector. Insequence, a thin layer of fibroin nanofibers, an intermixedlayer of fibroin and PCL nanofibers, and a thick layer of PCLelectrospun fibers were deposited on the grounded cylindricalmetallic collector. Each solution (fibroin or PCL solution) wasstored in a separate 5-mL syringe that served as a chargedspinneret. The syringe was fitted with a blunt-ended needleand connected to PTFE tubing. Each spinneret were placed atthe opposite side of the rotating mandrel, perpendicularlyoriented with respect to the principal axis of the collector,and connected to a separate high voltage power supply.Both spinnerets moved in concert over a 15-cm path witha speed of 5m/s along the mandrel under the control of alinear motorized stage (EZS3D025-C Oriental Motor ItaliaS.r.l., Milan, Italy). The feed rates of the two solutions werevaried independently between 0 and an optimal flow rateover the deposition time via two different syringe pumps.

Using single-polymer electrospinning processes, electro-spinning parameters were optimized for regenerated fibroinand PCL (Table I). These fabrication parameters were usedto produce fibroin-PCL composite scaffolds. Using the dou-ble electrospinning system described above, the silk-fibroin

solution stored in syringe A was first electrospun on the col-lector at a feed rate equal to the optimal value of 7 lL/min,while the PCL solution stored in syringe B were kept static.Thus, a thin layer of fibroin nanofibers was obtained. After 5min, the PCL solution started to be electrospun and depos-ited onto the collector with a low feed rate of 1 lL/min.This was done simultaneously with the electrospinning anddeposition of fibroin. During the next 10 min, the feed rateof the PCL solution was gradually adjusted to the optimalvalue of 15 lL/min, while the feed rate of the fibroinsolution were gradually reduced to zero. As a result, anintermixed interlayer of fibroin and PCL nanofibers wasdeposited on the top of the thin fibroin layer [Figure 1(b)].Finally, electrospinning of the PCL solution was conductedfor another 30–40 min, in order to obtain a thick PCL bulklayer. The obtained electrospun tubular constructs were cutalong the longitudinal axis, resulting in a smooth flat sheetwith a dimension of 6 " 12 cm2 and thickness approxi-mately in the range of 90–120 lm.

Post-treatment of scaffold materials containing fibroin. Awater/methanol mixture with 9:1 ratio was used for fibroinstabilization and detachment by dissolving PEO. Then, fi-broin was rinsed with DI water and soaked overnight in DIwater to eliminate solvent residuals.

Characterizations of the scaffold materials2.4.1 Scanning electron microscopy imaging. Nonwovenelectrospun meshes were imaged with scanning electronmicroscopy (SEM). To investigate net morphology, sampleswere mounted on aluminium stubs and sputter coated withgold. Observations were made at 10 kV by using a Stereo-scan 200 SEM (Cambridge Instrument Company, CambridgeU.K.). For nanofiber diameter analysis, three images weretaken in different locations of each sample, and roughly 100fiber diameters were measured and averaged using ImageJ, an image analysis software. Results were presented asmean 6 standard deviation.

FTIR analysisElectrospun mats were analyzed by FTIR in attenuate totalreflectance (ATR) mode with a Perkin Elmer Spectrum OneSpectrophotometer equipped with a diamond probe. In allcases 64 scans at a resolution of 2 cm#1 were used toobtain the spectra.

Thermal analysisDifferential scanning calorimetry (DSC) was performed on10 mg of material in an open crucible. The analysis wasperformed with a TA2920 calorimeter under flushing

FIGURE 1. Schematic illustrations of the double electrospinning systemand of the PCL-fibroin multilayered material. [Color figure can beviewed in the online issue, which is available at wileyonlinelibrary.com.]

TABLE I. Optimized Electrospinning Parameters

Solvent Formic acid Solvent DCM/DMF (3:1)

Concentration 10 wt % Concentration 9 wt %Voltage 22 kV Voltage 28 kVWork distance 12 cm Work distance 22 cmFeed rate 7 lL/min Feed rate 15 lL/min

278 BONANI ET AL. ANISOTROPIC BIOMECHANICAL PROPERTIES

nitrogen (100 mL/min). A sample was first cooled down to#100!C at the cooling rate of 50!C/min, and was held atthis temperature for 5 min. Subsequently, the first scan wasperformed by heating the sample up to 100!C with theheating rate of 10!C/min. This was followed by quenchingthe sample to #100!C. The second heating scan was carriedout by heating the sample from #100 to 315!C with theheating rate of 10!C/min and obtaining the thermogram.The melting peaks of PCL and PCL/fibroin samples werequantified by integration. Degree of crystallinity vc of PCLwas calculated using vc $ 100 " DHm/DHm

!, where DHm!

was the theoretical heat of fusion for PCL with 100%crystallinity equal to 135 J/g.

Mechanical characterizationMechanical properties were evaluated with uniaxial tensiletests on rectangular specimens (30 " 5 mm2 and 15 " 3mm2) cut along the circumferential (or transversal) direc-tion and the longitudinal direction. Silk-fibroin nets weretested in a wet condition as well as in a dry condition. Thefibroin nets were stored in water for three days at 4!Cbefore testing. For each specimen, three thickness measure-ments were taken using an electronic digital micrometerand averaged; three width measurements were taken usinga custom digital caliper and averaged. Thickness of thespecimens was in the range of 40–50 lm for fibroin electro-spun mats and 100–120 lm for PCL and compositesamples. Mechanical tests were performed with an electro-mechanical Instron 4502 (Instrom, Canton, MA) universaltensile testing machine equipped with a 100 N load cell.Samples were first preloaded to 0.10 N, and the gaugelength was recorded. The tensile test was carried to failureat a rate of 3 mm/min for fibroin samples and 10 mm/minfor PCL samples and multilayered samples. Elastic moduluswas calculated from the slope of the linear portion of theresultant stress-strain curve; maximum stress and failuredeformation were also calculated from the measurementdata. Four to five specimens were tested for each sample.The calculated values were averaged and reported as mean6 standard deviation. Students’ t-test was carried out forstatistic analysis of the data. A p value less than 0.05 wasconsidered statistically different; a p value less than 0.01was considered statistically very different.

Cell characterizationThe endothelial cells used for characterization were cellsisolated from proximal bovine pulmonary arteries and sub-cultured. Cells between passages three and six were used.Cells were cultured in Dulbecco’s Modified Eagle’s Medium(from Sigma-Aldrich) supplemented with 10% FBS, 2%L-Glutamine, and 1% penicillin-streptomycin at 37!C and5% CO2 in the cell culture incubator. Circular electrospunnets (diameter of 1.5 cm) were sterilized in 70% ethanol,allowed to dry and briefly exposed to UV light before seed-ing. Cells were cultured to 90% confluency in the cultureflask, detached, and then seeded on the fibroin side with1.0 mL of cell suspension per membrane at the concentra-tion of 200,000 cells/mL. Culture medium was changed

every other day. After four days of culture, the cell-seededmembranes were fixed with 4% formalin and stored in PBSat 4!C. To prepare samples for SEM examination, electro-spun nets were washed two to three times with distilledwater, exposed to different grades of ethanol for 10 min.Finally, they were air dried under laminar flow hood.Samples were sputter coated with gold and the morphologywas observed under SEM (JEOL 6480LV) at an acceleratingvoltage of 10 kV.

RESULTS

Fibroin and PCL electrospun materials produced by asingle-polymer electrospinning processWe have established single-polymer electrospinning pro-cesses to fabricate nanofibrous materials made of fibroinand PCL, respectively. The process parameters for each typeof material have been optimized using a flat collector in theelectrospinning system, and results are listed in Table I.SEM images (Figure 2) demonstrate the microscopic

FIGURE 2. SEM imaging graphs of silk-fibroin nanofibers (a) and PCLnanofibers (b) (scale bar $ 10 lm). At a higher magnification, detailsof fiber morphology are shown (scale bar $ 3 lm). The electrospin-ning processes were conducted on a flat aluminium collector with RH$ 70% and optimized electrospinning parameters listed in Table 1.



morphology of nanofibrous fibroin nets and PCL netselectrospun with the optimized parameters. In both nets,the nanofibers appear to have a rounded cross-section andregular shape, with diameters in the nanometric range. Theaverage diameters of fibroin and PCL nanofibers are 192 679 nm and 421 6 105 nm, respectively. Because severalstudies have reported the electrospinning process of PCLstructures,26,27 the optimization of PCL electrospinningparameters is not detailed here. Parametric studies on thefibroin electrospinning process are presented below becauseof its unique setup. In addition, using a rotational cylindricalmandrel, we have developed the electrospinning processesfor tubular structured fibroin and PCL, respectively. Para-metric studies for these electrospinning processes, in partic-ular, the effects of rotation speed on anisotropic mechanicalproperties of PCL nets and silk-fibroin nets, respectively,are presented.

Fibroin nets: parametric studies on nanofiber diameter.Four electrospinning parameters, namely voltage, solutionconcentration, feed rate and relatively humidity, have beenvaried to investigate their influences on the fibroin fiber di-ameter. The electrospun fibers usually show diameters rang-ing from tens of nanometers to one micron. It is found thatapplied electric voltage in the tested range does not signifi-cantly influence the diameter of silk-fibroin nanofibers [Fig-ure 3(a)], while the solution concentration plays an impor-tant role in determining the fiber diameter. No continuousdefect-free fibers have been obtained with the fibroin solu-tion at a concentration of 8% or lower in formic acid. Whenthe solution concentration is 10% or higher, the mean fiberdiameter increase exponentially with concentration; the

mean fiber diameter increases from 192 nm at 10% to 325nm at 14% [Figure 3(b)]. With our fabrication setup, it isnot possible to obtain fibroin solutions in formic acid at aconcentration higher than 14%. Another parameter, the feedrate at which the solution is forced out of the spinneret,does not significantly influence the mean fiber diameterwhen the feed rate is lower than 7 lL/min. Higher feedrates result in larger mean diameters [Figure 3(c)]. Further-more, environmental relative humidity (RH) shows a signifi-cant effect on the fiber diameter [Figure 3(d)]. Electrospin-ning in a relatively humid condition (RH $ 70%) results inmuch larger fibroin fibers compared with that in a lesshumid condition (RH $ 45%). Using the single-polymerelectrospinning process with optimized parameters (Table I)and a flat collector, fibroin nanofibers are characterized byan average diameter of 192 nm with a narrow normal-likedistribution (Figure 4). And the resultant electrospun netscontain a uniform array of randomly oriented fibers withsmooth rounded surface.

Fibroin nets: mechanical properties in a hydratedcondition. Silk-fibroin nanofiber scaffolds were prepared byelectrospinning fibers on a rotating cylindrical collectorwith a 20 mm diameter at varied rotation speeds including100, 250, 500, 1000, 1500, and 2000 rpm. As a reference,electrospun samples for 0 rpm were prepared by using arectangular collector with the same projected area as thecylindrical collector. Tensile tests were performed on thesamples cut from the tubular constructs in the longitudinaldirection and in the circumferential direction, respectively.Samples of a similar size from the rectangular scaffoldmaterials were used as the reference (0 rpm). Figure 5

FIGURE 3. Effects of electrospinning process parameters on the mean fiber diameter of electrospun silk-fibroin: (a) applied voltage; (b) solutionconcentration; (c) feed rate; and (d) environmental relative humidity (R.H.). Effects of R.H were studied at different electrospinning voltages:20 kV, 24 kV, and 28 kV.


demonstrates that the mechanical properties, elastic modu-lus and maximum strength, change as function of therotation speed of the mandrel. The samples collected at ahigh rotation speed, such as 500 rpm and 2000 rpm, showhigher elastic modulus and mechanical strength in thecircumferential direction. On the contrary, the samplescollected at a low rotation speed, such as 100 rpm, andthose collected from a static rectangular collector (0 rpm)show higher elastic modulus and mechanical strength in thelongitudinal direction. According to the data, an inversionpoint for the anisotropy of mechanical properties is

estimated to be around 200 rpm. However, at several rota-tion speeds, no statistically significant differences betweenthe mechanical properties in the two directions are found.Results also show that the strengths of fibroin nanofiberscaffolds fabricated in various conditions were always low—no higher than 1.7 MPa.

PCL anisotropic mechanical properties. PCL nanofiberscaffolds were prepared by electrospinning fibers on a rotat-ing cylindrical collector with a diameter of 20 mm at variedrotation speeds, including 100, 250, 500, 1000, 1500, and2000 rpm. Similar as fibroin, electrospun PCL samples for 0rpm were prepared by using a static rectangular collectorwith the same projected area as the cylindrical collector.Samples for mechanical testing were also collected in bothlongitudinal and circumferential directions. It is found thatthe mechanical properties of PCL samples show more aniso-tropic behaviors than fibroin samples. The anisotropy isalso dependent on the rotation speed of the mandrel collec-tor (Figure 6). The samples collected at a low rotationspeed, such as 0, 100, 250, 500, and 1000 rpm, show higherelastic modulus and ultimate strength in the longitudinaldirection. The samples collected at a high rotation speed,such as 1500 and 2000 rpm, show higher elastic modulusand ultimate strength in the circumferential direction. Theestimated inversion point for the anisotropy is between1000 and 1500 rpm.

FIGURE 4. Frequency distribution of the fiber diameter of silk-fibroinnets electrospun with optimized parameters shown in Table 1.

FIGURE 5. Elastic modulus and ultimate strength of silk-fibroin elec-trospun nets produced with a rotating collector. These properties inboth longitudinal and circumferential directions change with the rota-tion speed. Mechanical properties of the sample in the longitudinaldirection are significantly different from those in the circumferentialdirection (p < 0.05).

FIGURE 6. Elastic modulus and ultimate strength of PCL electrospunnets produced with a rotating collector. These properties in both lon-gitudinal and circumferential directions change with the rotationspeed. The mechanical properties of the sample in the longitudinaldirection are significantly different from those in the circumferentialdirection (p < 0.05 or p < 0.01).



Mechanical properties of small diameter constructs. PCLtubular constructs with a small diameter were preparedby electrospinning fibers on a cylindrical collector with adiameter of 4.96 mm rotating at 100 rpm. Specimens(15 " 3 mm2) cut along the longitudinal and circumferen-tial directions confirm the anisotropic mechanical behaviorof the electrospun materials. In longitudinal direction, anelastic modulus of 11.82 6 0.55 MPa and an ultimatestrength of 7.25 6 0.71 MPa were obtained; in the circum-ferential direction, the elastic modulus and ultimate strengthwere 4.04 6 1.17 MPa and 3.55 6 0.54 MPa, respectively.Thus, the mechanical anisotropic index (longitudinal-to-circumferential ratio of elastic modulus) results equal to 2.92.

Effect of collector shape. To test the assumption thatthe anisotropic mechanical properties are related to thegeometrical shape of the collector, we have designed a

series of collectors which are characterized by differentwidth-to-length aspect ratios (Figure 7). It was found thatnets electrospun on a slender rectangular collector withhigher width-to-length ratio present higher modulus andstrength along the length compared with those electrospunon a collector with lower width-to-length ratio. For example,for a width-to-length aspect ratio of 6, a mechanicalanisotropic index of 2.27 was found

Multilayered PCL-fibroin scaffold produced by a doubleelectrospinning processChemical and thermal characterizations of the structure. PCLand silk-fibroin have been electrospun on a cylindrical collec-tor rotating at a constant velocity to form a compositescaffold. With the new double-electrospinning apparatusdescribed above, a multilayered electrospun structure isformed and consists of an ultrathin layer of fibroin nanofib-ers, an interlayer of mixed nanofibers of fibroin and PCL,and a thick layer of PCL nanofibers. FT-IR analyses of thetwo opposite surfaces confirm that the layers are completelydifferent in the composition (Figure 8). The ATR FT-IRspectra show the presence of pure PCL on one side (calledPCL side) and fibroin on the other side (called fibroin side).Additionally, the shoulder at 1730 cm#1 in the FT-IR spec-trum of the fibroin side revealed a limited presence of PCL.

Figure 9 compares the DSC curve of a PCL-fibroin multi-layered material with that of a pure electrospun PCL mate-rial. In the DSC analysis, the presence of fibroin is revealedby a small endothermic peak around 280!C, which is corre-sponding to thermal degradation of silk-fibroin. It is knownfrom previous results that the degradation peak area for apure electrospun fibroin material is in the range of230–250 J/g. Thus, using the area of the small peak shownin the composite scaffold (around 10–12 J/g), the overallcontent of fibroin inside the multilayered scaffold can beestimated to be around 5% in weight. The degrees ofcrystallinity of PCL for PCL and PCL/fibroin samples areequal to 40.4 % and 37.5 %, respectively.

FIGURE 7. Effect of collector shape on the mechanical property ofPCL electrospun material. Elastic modulus along the two principaldirections of the electrospun nets is reported as function of thelength/width ratio of the collector (p < 0.05).

FIGURE 8. ATR FT-IR analysis of the scaffold shows completely differ-ent compositions on the two surfaces. PCL side shows the character-istic spectrum of PCL, and fibroin side shows the characteristicspectrum of prevalent b-sheet fibroin and a small contamination ofPCL due to the limited thickness of the fibroin layer.

FIGURE 9. DSC thermographs for PCL/fibroin multilayered material,pure PCL, and pure Fibroin. The small endothermic peak at 280!C,which is common to fibroin and PCL/fibroin multilayered material, isattributed to the thermal degradation of silk fibroin.


Anisotropic mechanical properties of the multilayeredstructure. The mechanical properties of nanofibrousmultilayered PCL-fibroin scaffolds have been studied andevaluated in comparison to the mechanical properties of

pure PCL scaffolds prepared in the same conditions[Figure 10(a)]. No significant differences in elastic modulusor in ultimate strength (data not shown) are found betweenmultilayered PCL-fibroin scaffolds and pure PCL scaffolds inboth circumferential and longitudinal directions. This isfound on the samples collected at a low rotation speed(150 rpm) and on those collected at a high rotation speed(1500 rpm). Therefore, fibroin does not significantlycontribute to the mechanical property of the multilayeredcomposite scaffold. This is also shown by representativestress-strain curves for multilayered PCL-fibroin materialand pure PCL electrospun material using specimens cut inthe circumferential direction [Figure 10(b)]. These curvesalso show that the mechanical property of fibroin electro-spun material is dependent on the humidity; it is muchstiffer and stronger in a dry condition than it is in ahydrated condition.

Endothelial cell characterization.. Endothelial cell culturetest was performed on flat electrospun constructs usingprimary arterial endothelial cells (Figure 11). A low concen-trated cell suspension was seeded on the two differentsurfaces of the multilayered electrospun structure. Cellmorphology and adhesion site were examined after fourdays of culture. Endothelial cells seeded on the fibroin sideresulted in a highly spread morphology, protruding cyto-plasmic interconnections between the cells along a largernumber of electrospun fibers.

DISCUSSION

This study has reported the development of electrospinningprocesses of silk-fibroin, PCL, and PCL-fibroin multilayercomposite for engineered vascular construct. Using a single-spinneret electrospinning system with a flat collector, wehave optimized electrospinning parameters to obtain defect-free, uniform nanofibers from pure PCL and puresilk-fibroin, respectively. In particular, the effects of appliedvoltage, protein concentration, feed rate and environmentalhumidity on the fibroin fiber diameter are demonstrated.Using a rotating cylindrical collector, anisotropic mechanicalbehaviors of these pure nanofiber materials are obtainedand the anisotropy is found to be dependent on the rotationspeed of the collector. In addition, we have developed a new

FIGURE 10. (a) Elastic modulus of multilayered PCL-fibroin materialsand pure PCL materials. The material fabrication used a cylindricalcollector rotating at a low speed (150 rpm) and a high speed (1500rpm). All the tests were performed on the circumferential direction‘‘C’’ and the longitudinal direction ‘‘L’’ of the samples. No significantdifferences in elastic modulus were found (p > 0.05). (b) Representa-tive stress-strain curves of hydrated fibroin, dry fibroin, PCL, andPCL-fibroin electrospun nets. The material fabrication used a cylindri-cal collector rotating at a high speed (1500 rpm). All the tests wereperformed on the circumferential direction of the tubular scaffolds.

FIGURE 11. SEM images of bovine pulmonary artery endothelial cells seeded on the fibroin side of the multilayered structure (4 days in culture).



double-electrospinning system, and have achieved a promis-ing combination of mechanical property and biologicalproperty with a biohybrid nanofibrous scaffold made fromPCL and silk-fibroin.

Electrospinning of silk fibroinA major difference in fibroin electrospinning and characteri-zation presented here from previous studies,28,29 is that wehave taken the environmental relative humidity (RH) andthe hydration state of a fibroin sample into consideration.We have shown that the diameter of fibers electrospun at45% RH were about half of that at 70% RH [Figure 4(d)]. Itis likely because water molecules reduce the electric chargedensity on the developing fibers, thus reducing the drivingforce for fiber elongation and diameter reduction. Also, asingle fiber can be stretched more in a dry environmentbecause the viscosity remains low for a longer time, aneffect equivalent to extending the distance from the needle,while a fiber in a more humid environment becomes solidfaster and experiences a lower degree of stretching. In addi-tion, we have demonstrated the difference in mechanicalproperties between dry and hydrated fibroin nets [Figure10(b)]. It is found that hydration highly reduces stiffnessand strength of the electrospun material. Because thescaffold is often used in the hydrated state, the majority ofmechanical characterizations performed here are based onhydrated samples. The ultimate tensile strengths reportedin this study are comparable to the values reported bySoffer et al.30 who electrospun hydrated fibroin nets from aPEO/fibroin water solution, but the elastic modulusreported here are much higher, ranging from 5.32 to 12.11MPa. The difference is likely attributed to a much highertesting rate (3 mm/min compared to 0.2 mm/min) and thepreconditioning cycles used in this study.

Effect of the rotation speed and the collector shape onanisotropic mechanical propertiesThis study have shown that varying rotation speed leads tochanges in the material mechanical anisotropy index whichis defined as the ratio of the mechanical property in thelongitudinal direction to that in the circumferential direc-tion. With the rotation speed varying from 0 to 2000 rpm,anisotropy indices of elastic modulus range from 1.7 to 0.35for PCL material, and anisotropy indices range from 1.2 to0.70 for fibroin material. PCL shows more gradual changesin the anisotropy index with the rotation speed, and a widerrange of anisotropy indices is achievable with PCL. In addi-tion, no significant differences are found between pure PCLnets and multilayered PCL-fibroin nets both at 150 rpm andat 500 rpm. This suggests that mechanical properties of aPCL-fibroin composite are similar to those of a pure PCLelectrospun material. This could be due to the small amountof fibroin (%5% in weight) in the composite constructaccording to the DSC analysis results. The dependence ofthe mechanical anisotropy index on the rotational speedcould be explained by the relationship between fiber align-ment and rotational speed, according to previous studieswhich showed that a rotational collector tended to align the

fibers along the circumferential direction at higher rotationspeeds.31 The nanofiber alignment in the circumferentialdirection happens when the tangential velocity at the collec-tor surface exceeds the velocity at which the nanofibersapproach the collector, particularly when the rotation speedbecomes higher. Additionally, many studies have shown astrong correlation between mechanical properties and fiberalignment in non-woven electrospun materials,32,33; progres-sive alignment of the fibers in one direction improveselastic modulus and tensile strength of the material in thesame direction.

It is also noticed in this study that at 0 rpm and atlower rotation speeds, both PCL and fibroin materials showhigher modulus and strength in the longitudinal directionthan in the circumferential direction. Because there is nomandrel rotation, it may involve the effect of collectorshape. In order to test the assumption that the anisotropicmechanical properties are related to the geometrical shapeof the collector, we have designed a series of collectorswhich are characterized by different width-to-length aspectratios (Figure 7). It was found that nets electrospun on aslender rectangular collector with higher width-to-lengthratio present higher modulus and strength along the lengthcompared with those electrospun on a collector with lowerwidth-to-length ratio. The distortion of the electric field inthe air gap might induce the alignment of fibers along theprevalent geometrical direction and thus higher mechanicalproperties along that direction. The collector shape effect onthe mechanical anisotropic index is also important at lowerrotation speeds when the effect of mandrel rotation is negli-gible. The inversion point, where the anisotropic index isequivalent to 1, suggests the balance between the collectorshape effect and the rotation speed effect. It is about 1200rpm for PCL and 150 rpm for fibroin.

At low rotation speed of the mandrel the material in thelongitudinal direction is stronger and stiffer than in circum-ferential direction. This property is desired for vasculargrafts. Therefore, we also produced constructs with asmaller diameter (4.76 mm) at a low rotation speed of 100RPM. After mechanically testing the construct in longitudi-nal and in circumferential directions we found that theanisotropic behavior was also demonstrated by small diame-ter constructs. In particular, elastic modulus and ultimatestrength in the longitudinal direction are higher than therespective values in the circumferential direction. The longi-tudinal-to-circumferential ratio was 2.92.

Double electrospinning systemDouble electrospinning combines the advantageous proper-ties of two types of materials, but more importantly, it betterintegrates their nanofibers compared with a simple overlap-ping of nanofibers. We have shown that the multilayeredstructure resulted from double electrospinning improvesbiological properties of PCL as endothelial adhesion potentialon the scaffold surface increases with a natural cell-condu-cive polymer (fibroin), while it retains superior mechanicalproperties of the synthetic slow degradable polymer (PCL).Furthermore, as shown in Figure 9(b), the stress-strain


behaviors of fibroin and PCL are quite different, which resultin completely different behaviors during deformation. As nostrong adhesion exists at the PCL and fibroin interface, asimple overlapping of a PCL layer and a fibroin layer leadsto delamination of the construct during deformation. There-fore, the fundamental importance of the double electrospin-ning system resides in the possibility to produce a mixedfiber interlayer. The intermixed region acts like an activeinterphase that transmits interfacial forces and avoidsdelamination. Different from the other recently developeddouble electrospinning system by Baker et al.,34 the systemestablished in this study designs two charge needles, fromwhich the polymers solutions are ejected, to move concur-rently along the rotating mandrel, thus resulting in a moreeffective mixing of the different materials. In addition, thereare no charged shields placed perpendicularly to the man-drel, thus preventing any modifications of the electric field inthe region between the charge spinnerets and the collector.

Application of the scaffolds in tissue engineeringMechanical property and surface biological property areboth important for vascular scaffolding materials. To repro-duce anisotropic mechanical property of arteries, engineeredvascular scaffolds need sufficient radial compliance andrelatively higher longitudinal stiffness (anisotropy index> 1). Thus electrospinning processes at lower rotationspeeds of the mandrel are likely useful to produce theseanisotropic scaffolds, and changing the mandrel rotationspeed makes the anisotropy tunable, particularly with PCLbulk materials. Using a multilayer structure with bulk PCLand limited amount of fibroin on the surface, we can obtainnot only an optimized combination of anisotropic mechani-cal property, but also surface properties for cells. Comparedwith PCL, a fibroin-based matrix better supports endothelialcell growth due to the favorable biological properties of thesilk protein. Besides the composition, the net morphology isalso important for the formation of effective endotheliallayer. Other studies also demonstrated that three-dimen-sional silk-fibroin nets derived from the cocoons of the silkworm, Bombyx mori, support the attachment, spread andgrowth of a variety of human cell types of diverse tissue ori-gins;35 in particular, it has been reported that the regener-ated silk-fibroin maintains normal cell phenotypes and func-tions of primary human macro- and micro-vascularendothelial cells as well as endothelial cell lines,36 and hasanti-thrombogenic potential.20

CONCLUSION

We have demonstrated a double-electrospinning techniqueto produce a robust tubular scaffold with well-integratednanofibrous structure, anisotropic mechanical behavior, andendothelial-conducive surface, for vascular tissue engineer-ing. A thick bulk PCL mesh of nanofibers, an intermediateintermixed fibrous layer, and a thin layer of silk-fibroinnanofibers have been combined to form a multilayeredstructure which is stable against mechanical delamination.The structure can be stiffer in the longitudinal direction and

more compliant in the circumferential direction, and reachup to 2.5 in the longitudinal-to-circumferential ratio in thisstudy. The combination of the collector shape and thecollector rotation allows us to produce a tubular structurewith tunable anisotropic mechanical properties. The phe-nomenon has been extensively demonstrated in the case ofelectrospinning on a 20 mm mandrel, while can be generallyapplied. In fact, samples obtained with a 4.96 mm mandrelrotating at 100 rpm confirm the anisotropic behavior reach-ing a longitudinal-to-circumferential ratio higher than 2.9. Inaddition to vascular scaffolds, the system and techniquedeveloped in this study will be important for many othertissue engineering applications as well.

ACKNOWLEDGMENTS

The authors would like to acknowledge Dmitry Belchenko forproviding assistance in cell characterization and CVP group forproviding bovine pulmonary arterial endothelial cells. Thiswork was performed in part at the University of Colorado’sNanomaterials Characterization Facility at Boulder, CO.

REFERENCES1. Hutmacher D. Scaffold design and fabrication technologies for

engineering tissues—State of the art and future perspectives.J Biomater Sci-Polym Ed 2001;12:107–124.

2. Chen G, Ushida T, Tateishi T. Scaffold design for tissue engineer-ing. Macromol Biosci 2002;2:67–77.

3. Hutmacher D. Scaffolds in tissue engineering bone and cartilage.Biomaterials 2000;21:2529–2543.

4. Conte M. The ideal small arterial substitute: A search for the HolyGrail? Faseb J 1998;12:43–45.

5. Zilla P, Bezuidenhout D, Human P. Prosthetic vascular grafts:Wrong models, wrong questions and no healing. Biomaterials2007;28:5009–5027.

6. Ratcliffe A. Tissue engineering of vascular grafts. Matrix Biol2000;19:353–357.

7. Fung YC. Biomechanics: Mechanical Properties of Living Tissues,2nd ed. Springer; 1993.

8. Vossoughi J. Book review: Data book on mechanical propertiesof living cells, tissues and organs, edited by H. Abe, K. Hayashi,and M. Sato. Ann Biomed Eng 1998;26:735.

9. Xue L, Greisler H. Biomaterials in the development and future ofvascular grafts. J Vasc Surg 2003;37:472–480.

10. Haruguchi H, Teraoka S. Intimal hyperplasia and hemodynamicfactors in arterial bypass and arteriovenous grafts: A review.J Artif Organs 2003;6:227–235.

11. Li W, Laurencin C, Caterson E, Tuan R, Ko F. Electrospun nanofi-brous structure: A novel scaffold for tissue engineering. J BiomedMater Res 2002;60:613–621.

12. Yoshimoto H, Shin Y, Terai H, Vacanti J. A biodegradable nano-fiber scaffold by electrospinning and its potential for bone tissueengineering. Biomaterials 2003;24:2077–2082.

13. Pham Q, Sharma U, Mikos A. Electrospinning of polymeric nano-fibers for tissue engineering applications: A review. Tissue Eng2006;12:1197–1211.

14. Mo X, Weber H. Electrospinning P(LLA-CL) nanofiber: A tubularscaffold fabrication with circumferential alignment. MacromolSymp 2004;217:413–416.

15. Xu C, Inai R, Kotaki M, Ramakrishna S. Electrospun nanofiberfabrication as synthetic extracellular matrix and its potential forvascular tissue engineering. Tissue Eng 2004;10(7–8):1160–1168.

16. Burger C, Hsiao B, Chu B. Nanofibrous materials and their appli-cations. Ann Rev Mater Res 2006;36:333–368.

17. Altman G, Diaz F, Jakuba C, Calabro T, Horan R, Chen J, et al.Silk-based biomaterials. Biomaterials 2003;24:401–416.



18. Unger R, Sartoris A, Peters K, Motta A, Migliaresi C, Kunkel M,et al. Tissue-like self-assembly in cocultures of endothelial cellsand osteoblasts and the formation of microcapillary-like struc-tures on three-dimensional porous biomaterials. Biomaterials2007;28:3965–3976.

19. Kim H, Kim U, Leisk G, Bayan C, Georgakoudi I, Kaplan D. Boneregeneration on macroporous aqueous-derived silk 3-D scaffolds.Macromol Biosci 2007;7:643–655.

20. Zhang X, Baughman C, Kaplan D. In vitro evaluation of electro-spun silk fibroin scaffolds for vascular cell growth. Biomaterials2008;29:2217–2227.

21. Bondar B, Fuchs S, Motta A, Migliaresi C, Kirkpatrick C. Function-ality of endothelial cells on silk fibroin nets: Comparative study ofmicro- and nanometric fibre size. Biomaterials 2008;29:561–572.

22. Hutmacher D, Goh J, Teoh S. An introduction to biodegradablematerials for tissue engineering applications. Ann Acad MedSingapore 2001;30:183–191.

23. Li W, Danielson K, Alexander P, Tuan R. Biological response ofchondrocytes cultured in three-dimensional nanofibrous poly(ep-silon-caprolactone) scaffolds. J Biomed Mater Res Part A 2003;67:1105–1114.

24. Kweon H, Yoo M, Park I, Kim T, Lee H, Lee H, et al. A noveldegradable polycaprolactone networks for tissue engineering.Biomaterials 2003;24:801–808.

25. Valluzzi R, He S, Gido S, Kaplan D. Bombyx mori silk fibroinliquid crystallinity and crystallization at aqueous fibroin-organicsolvent interfaces. Int J Biol Macromol 1999;24(2–3):227–236.

26. Pham Q, Sharma U, Mikos A. Electrospun poly(epsilon-caprolac-tone) microfiber and multilayer nanofiber/microfiber scaffolds:Characterization of scaffolds and measurement of cellular infiltra-tion. Biomacromolecules 2006;7:2796–2805.

27. Luong-Van E, Grondahl L, Chua K, Leong K, Nurcombe V, Cool S.Controlled release of heparin from poly(epsilon-caprolactone)electrospun fibers. Biomaterials 2006;27:2042–2050.

28. Sukigara S, Gandhi M, Ayutsede J, Micklus M, Ko F. Re-generation of Bombyx mori silk by electrospinning, part 1. Proc-essing parameters and geometric properties. Polymer 2003;44:5721–5727.

29. Sukigara S, Gandhi M, Ayutsede J, Micklus M, Ko F. Regenerationof Bombyx mori silk by electrospinning, Part 2. Process optimiza-tion and empirical modeling using response surface methodol-ogy. Polymer 2004;45:3701–3708.

30. Soffer L, Wang X, Mang X, Kluge J, Dorfmann L, Kaplan D, et al.Silk-based electrospun tubular scaffolds for tissue-engineeredvascular grafts. J Biomater Sci-Polym Ed 2008;19:653–664.

31. Thomas V, Jose M, Chowdhury S, Sullivan J, Dean D, Vohra Y.Mechano-morphological studies of aligned nanofibrous scaffoldsof polycaprolactone fabricated by electrospinning. J BiomaterSci-Polym Ed 2006;17:969–984.

32. Li W, Mauck R, Cooper J, Yuan X, Tuan R. Engineering controlla-ble anisotropy in electrospun biodegradable nanofibrous scaf-folds for musculoskeletal tissue engineering. J Biomechan 2007;40:1686–1693.

33. Kenawy E, Layman JM, Watkins JR, Bowlin GL, Matthews JA,Simpson DG, et al. Electrospinning of poly(ethylene-co-vinylalcohol) fibers. Biomaterials 2003;24:907–913.

34. Baker B, Gee A, Metter R, Nathan A, Marklein R, Burdick J, et al.The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrifi-cial fibers. Biomaterials 2008;29:2348–2358.

35. Unger R, Wolf M, Peters K, Motta A, Migliaresi C, Kirkpatrick C.Growth of human cells on a non-woven silk fibroin net:A potential for use in tissue engineering. Biomaterials 2004;25:1069–1075.

36. Unger R, Peters K, Wolf M, Motta A, Migliaresi C, Kirkpatrick C.Endothelialization of a non-woven silk fibroin net for use in tissueengineering: Growth and gene regulation of hunian endothelialcells. Biomaterials 2004;25:5137–5146.


Biohybrid nanofiber constructs with anisotropic biomechanical properties

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