andrea r. tan et al- electrospinning of photocrosslinked and degradable fibrous scaffolds

8/3/2019 Andrea R. Tan et al- Electrospinning of photocrosslinked and degradable fibrous scaffolds

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Electrospinning of photocrosslinked and degradable

fibrous scaffolds

Andrea R. Tan,1 Jamie L. Ifkovits,1 Brendon M. Baker,1,2 Darren M. Brey,1 Robert L. Mauck,1,2

Jason A. Burdick11Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 191042McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, University of Pennsylvania,Philadelphia, Pennsylvania 19104

Received 23 July 2007; revised 27 September 2007; accepted 22 October 2007Published online 6 February 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31853

Abstract: Electrospun fibrous scaffolds are being devel-oped for the engineering of numerous tissues. Advantages ofelectrospun scaffolds include the similarity in fiber diameterto elements of the native extracellular matrix and the abilityto align fibers within the scaffold to control and direct cellu-lar interactions and matrix deposition. To further expand therange of properties available in fibrous scaffolds, we devel-oped a process to electrospin photocrosslinkable macromersfrom a library of multifunctional poly(b-amino ester)s. Inthis study, we utilized one macromer (A6) from this libraryfor initial examination of fibrous scaffold formation. A car-rier polymer [poly(ethylene oxide) (PEO)] was used for fiberformation because of limitations in electrospinning A6 alone.Various ratios of A6 and PEO were successfully electrospunand influenced the scaffold fiber diameter and appearance.When electrospun with a photoinitiator and exposed to light,the macromers crosslinked rapidly to high double bond con-versions and fibrous scaffolds displayed higher elastic mod-

uli compared to uncrosslinked scaffolds. When these fiberswere deposited onto a rotating mandrel and crosslinked,organized fibrous scaffolds were obtained, which possessedhigher moduli (4-fold) in the fiber direction than perpen-dicular to the fiber direction, as well as higher moduli (12-fold) than that of nonaligned crosslinked scaffolds. Withexposure to water, a significant mass loss and a decrease inmechanical properties were observed, correlating to a rapidinitial loss of PEO which reached an equilibrium after7 days. Overall, these results present a process that allowsfor formation of fibrous scaffolds from a wide variety of pos-sible photocrosslinkable macromers, increasing the diversityand range of properties achievable in fibrous scaffolds for tis-sue regeneration. 2008 Wiley Periodicals, Inc. J BiomedMater Res 87A: 10341043, 2008

Key words: polymers; biodegradable; fibrous scaffolds;electrospinning; poly(b-amino esters)

INTRODUCTION

Electrospun biodegradable scaffolds are a usefultool for the engineering of numerous tissues, particu-larly those of the musculoskeletal system.1 To fabri-cate such scaffolds, suspended droplets of a polymermelt solution are electrically charged until chargerepulsion overcomes the surface tension of the drop-let and a stable polymer jet is formed. As the jetelongates and solvent evaporates, an ultrathin poly-mer strand is formed and collected onto a groundedsurface.2 The fibers within the resulting scaffold

more closely mimic the size and structure of thenative extracellular matrix than other previouslyinvestigated scaffolds, which can lead to beneficialcellular interactions.3 Additionally, collection meth-odologies used in forming such scaffolds can be var-ied.4,5 For example, a rotating collection mandrel can

be deployed to generate scaffolds with a high degree

of fiber alignment. These structures may then serveas 3D micropatterns whose architecture mimics thatof many fiber-reinforced tissues.69

These nano- and micro-scale fibrous scaffolds havemany applications for tissue engineering. Our ownlong-term goal is to use such scaffolds for the repairof fiber-reinforced tissues of the musculoskeletal sys-tem. These tissues, including tendon, ligament, theannulus fibrosus region of the intervertebral disc, andthe knee meniscus, all function to transmit the highmechanical forces that arise with locomotion. Hall-marks of these dense fibrous tissues include a highcollagen content organized along a prevailing fiber

Correspondence to: J. A. Burdick; e-mail: [email protected]

Contract grant sponsors: University of PennsylvaniaUniversity Research Foundation, Merck UndergraduateResearch Scholarship (ART), Ashton Fellowship (JLI),Department of Education GAANN Fellowship (BMB)

2008 Wiley Periodicals, Inc.


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direction that is generally coincident with the princi-pal loading direction. This characteristic enables tis-sue function, which is significantly diminished fol-lowing injury and the formation of disorganized scartissue. Taking the meniscus as an example, there are1,500,000 arthroscopic surgical procedures of the

knee performed annually, and of these, more thanhalf are related to the meniscus fibrocartilage of theknee.10 Injury to, or loss of, the meniscus inhibits effi-cient load transfer in the knee and precipitates long-term joint changes, including osteoarthritis. Currenttreatments for meniscus damage (including partial ortotal meniscectomy) treat acute symptoms such aspain and joint locking,11,12 but do not restore functionto the knee. We have recently reported on the fabrica-tion of engineered meniscus constructs using a fiber-aligned electrospun scaffolding system, and haveshown that the scaffold architecture can promotefunctional maturation of constructs.7

A number of biodegradable polymers, such aspoly(a-hydroxy esters)13,14 and polyurethanes,15 aswell as natural polymers, such as collagen1618 andsilk fibroin,1620 have been electrospun into scaffoldsfor use in tissue regeneration applications. Each start-ing polymer possesses unique mechanical and degra-dation properties, allowing for a range of scaffoldproperties to be obtained. By carefully selecting thepolymer, one can control the initial mechanical prop-erties and degradation kinetics of the electrospun scaf-fold.14 For example, a recent study by Li et al. showedthat the electrospinning of six different poly(a-

hydroxy esters) resulted in scaffolds whose mechanicsand degradation depended on the choice of polymerfor scaffold fabrication.14 While existing polymersshow promise, it may be useful to further expand theavailable polymers used for electrospinning fibrousscaffolds to optimize cellular interactions, bulk prop-erties, and ultimately tissue regeneration.

To this end, we have recently synthesized a largelibrary of photopolymerizable and degradable macro-mers based on poly(b-amino esters) (PBAEs).21 Photo-crosslinkable PBAE macromers are easily synthesizedwith no byproducts, and macromer constituents areinexpensive and commercially available. When formed

as films, members of this library show a wide range ofphysical properties (mechanics and degradation),spanning at least two orders of magnitude.21 Further-more, it was recently shown that simple alterations inPBAE molecular weight can lead to dramatic changesin polymer mechanics, degradation, and cellular inter-actions.22 Such a range of properties in this library may

be useful in the optimization of fibrous scaffold prop-erties toward tissue regeneration applications.

The overall aim of this study was therefore toassess the feasibility of electrospinning photocros-slinkable macromers, using a candidate from thenovel PBAE library as an example. There are no cur-

rent techniques for the electrospinning of low molec-ular weight photocrosslinkable polymers, and thus,the novelty to this work is the development of newprocessing technology. Specifically, one macromerfrom the library was selected based on its favorable

bulk properties (e.g., slow degradation and viable

cellular interactions) and was electrospun into fibrous scaffolds. The formed scaffolds were charac-terized with respect to reaction behavior, mass loss,and degradation products, and both initial mechani-cal properties and those with degradation. An in-depth analysis of these scaffolds toward the regener-ation of a specific tissue is beyond the scope of thisreport, yet this study represents a significantadvance in the design of fibrous scaffolds for tissueregeneration since a wide range of radically cross-linked and biodegradable polymers can now be elec-trospun using this process. Importantly, these resultsshow the feasibility of electrospinning photocros-slinkable macromers and provide a foundation forfurther optimization of engineered fibrous tissues.

MATERIALS AND METHODS

Macromer synthesis and selection

PBAEs were synthesized by the conjugate addition ofprimary amines to diacrylates by mixing the liquid precur-sors and reacting overnight at 908C with stirring.21 For thesynthesis of macromer A6, diethylene glycol diacrylate (A,

Scientific Polymer Products) was mixed with isobutyl-amine (6, Sigma) in a 1.2:1 molar ratio. This notation coin-cides with our initial report on the PBAE library. The A6molecular weight was confirmed using 1H NMR (BrukerAdvance 360 MHz, Bruker, Billerica, MA). Polymer filmswere made by first adding the photoinitiator 2-dimethoxy-2-phenyl acetophenone (DMPA, Ciba-Geigy) to the macro-mer by dissolving DMPA in methylene chloride, mixingwith the macromer, and evaporating off the methylenechloride in a dessicator. The macromer/DMPA solutionwas injected between two glass slides with a 1-mm spacerand polymerized with exposure to ultraviolet light (BlakRay, 10 mW/cm2) for 10 min. For initial studies, 1-cm-diameter discs were punched from the slabs and degraded

in phosphate-buffered saline (PBS) at 378C and the massloss was recorded over 12 weeks.

For cellular interaction studies, films were prepared onthe bottom of tissue culture plates as previouslydescribed.22 Briefly, the macromer/initiator solution wasdissolved in ethanol at a 1:2 (w/v) ratio and polymerizedin 24-well plates under nitrogen purge with ultravioletlight for 10 min. Films were sterilized with exposure to agermicidal lamp in a laminar flow hood for 30 min andthen treated with PBS and media prior to cell seeding. Bo-vine mesenchymal stem cells (MSCs) were isolated fromthe tibial trabecular bone marrow of 36-month-old calvesand expanded as described in Ref. 23. MSCs were seededon the films at a density of 50,000 cells/well in DMEM

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supplemented with 10% fetal bovine serum and 13 peni-cillin/streptomycin/fungizone and cultured for either 24 hor 7 days. At these time points, cells were fixed in formalin-free fixative (Accustain, Sigma) for 25 min. The cells werepermeabilized with 0.25% Triton X-100 in PBS for 10 min,and nonspecific binding sites were blocked with 3% bovine

serum albumin and 0.1% Tween-20 in PBS for 15 min at378C. The fixed cells were stained for actin with TRITC-conjugated phalloidin (1 lg/mL in PBS, Sigma, St. Louis,MO) for 30 min at room temperature. The nuclei werethen stained with DAPI (1:2500, Sigma) for 5 min at roomtemperature. Films were rinsed three times with PBSbetween each of the previous steps and images were takenon a fluorescent microscope (Axiovert, Zeiss, Germany)with a digital camera (Axiovision, Zeiss). The total cellnumber was determined by counting nuclei in at least fiverandom fields on five individual films for each composi-tion at each time point. The area of the field was deter-mined using a Bright-Line hemacytometer.

Electrospinning of nanofibrous scaffolds andcharacterization

For electrospinning, macromer A6 was dissolved in 90%EtOH to obtain a final macromer concentration of 50%(w/v). DMPA was then added to attain a final DMPA solu-tion of 0.5 wt % of the mass of macromer. Poly(ethylene ox-ide) (PEO, 200 kDa) was introduced as a carrier polymer tofacilitate fiber formation during electrospinning. A 10% (w/v) solution of PEO was produced by dissolution in 90%EtOH, and the PEO and A6 solutions were mixed at variousratios. Electrospinning was carried out using a customapparatus consisting of a metal spinneret through whichthe polymer solution is expressed, a high voltage powersupply, and a grounded collecting surface.9 Spinneretswere 18-gauge blunt-ended needles and the solution flowrate was governed by gravimetric pressure. Solutions wereelectrospun at 13 kV over 20 cm and collected for 90 min ona stationary plate (for SEM and FTIR analysis) or for 12 hon a rotating mandrel at 10 m/s (for mass loss and tensiletesting). Nonaligned mats were also fabricated by spinningonto a stationary plate for 12 h (for tensile testing). The scaf-folds ranged in thickness from 0.6 to 1.4 mm, dependingon alignment and position on the mandrel.

Scaffolds were analyzed with scanning electron micros-copy (SEM, JEOL 6400, Penn Regional NanotechnologyFacility) before and after polymerization (365 nm wave-length, 5 mW/cm2). Fiber diameter was quantified usingthe length measurement tool in ImageJ (v1.38, NIH). Onehundred measurements were taken for each SEM imagefor the different PEO:A6 ratios. Double bond conversionwithin fibrous scaffolds was monitored with ultravioletlight exposure using Attenuated Total Internal Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR, Nico-let 6700, Thermo Fisher Scientific, Waltham, MA) and real-time monitoring of the acrylate peak (1630 cm21).

Mechanical properties and mass loss

Rectangular samples (5 3 25 mm2) were excised asstrips from electrospun sheets for mechanical testing. Uni-

axial tensile testing was performed on both untreated scaf-folds (uncrosslinked) and after sheets were crosslinkedwith ultraviolet light. Samples were taken from both nona-ligned and aligned sheets, and tested parallel and perpen-dicular to the prevailing fiber direction. Additionally,aligned samples were incubated in water for 1, 3, or

6 days, dried by lyophilization, and mechanically tested.Dried constructs were preconditioned with 10 sinusoidalcycles of 0.5% strain at 0.1 Hz and subsequently tested tofailure at a constant strain rate of 0.1%/s using a 5848Microtester equipped with a 50-N load cell (Instron, Can-ton, MA). The tensile modulus of the construct was calcu-lated from the linear region of the stressstrain curve (03%) and initial sample geometry.

Five-millimeter-diameter punches were excised andmass loss was determined after incubation in deionizedwater at 378C for 6 h and subsequently at daily intervalsover the period of a week. The fiber morphology was alsomonitored throughout the period of mass loss. The solublefraction of the bulk polymer was determined by immers-

ing the punches in methylene chloride overnight to allowunreacted macromer to swell from the network. After dry-ing, the soluble fraction was calculated as a percentage ofinitial mass loss and repeated daily until the daily incre-mental mass loss was negligible.

Additionally, 1H NMR was used to investigate theeluted products after incubation in deionized water. Thesupernatant at each mass loss time point was collected, ly-ophilized, and prepared for 1H NMR analysis by dissolv-ing each sample in deuterated chloroform. A 65:35 PEO:A6sample was also prepared for analysis. Each sample wasscanned and processing was completed using SpinWorks(University of Manitoba, Canada). Values are reported as aratio of the amount of PEO to A6 at the mass loss time

point to the ratio of the amount of PEO to A6 in the initialelectrospun solution.

Statistical analysis

Anova with Tukeys post hoc test was used to determinesignificant difference among groups, with p < 0.05. All val-ues are reported as the mean 6 the standard deviation.

RESULTS

Macromer synthesis and characterization

One macromer, termed A6, was selected from thePBAE library and synthesized by the step-growthpolymerization of the amine and diacrylate [Fig.1(A)]. End group analysis of the 1H NMR spectrawas used to determine the number average molecu-lar weight of A6 to be 1.34 kDa. After synthesis,macromer A6 was crosslinked into networks using afree-radical photoinitiated polymerization. A6 wasidentified as a potential candidate for tissue engi-neering applications because of its degradation pro-file and favorable cellular interactions. The degrada-tion profile shows gradual mass loss over 3 months

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[Fig. 1(B)] and is indicative of a bulk degradingpolymer. Although this does not span complete deg-radation, placement of networks in strong base leadsto complete degradation (data not shown).

The interaction of MSCs with films of A6 and a con-trol of tissue culture polystyrene (TCPS) was assessedand showed comparable adhesion after both 24 h and7 days. For instance, cell adhesion on networks fabri-cated from A6 was 98% and 80% the numbers ofcells on TCPS, after 24 h and 7 days, respectively. Theinteraction of MSCs with the films was also assessed

by actin and nuclear staining [Fig. 1(C,D)]. After7 days, a near confluent monolayer of cells wasobserved on both surfaces with actin fibers morepronounced in MSCs cultured on A6 than on TCPS.

Fibrous scaffold fabrication

Because of the desirable initial properties of A6(i.e., slow degradation, cytocompatibility), this mac-

romer was selected for electrospinning. However,initial attempts to spin A6 alone resulted in electro-spraying, whereby beads were formed, likely due tothe low molecular weight of A6. Thus, a process wasdeveloped in which A6 was electrospun with a highmolecular weight carrier polymer (i.e., PEO). A sche-

matic of the electrospinning process is shown in Fig-ure 2, where a polymer solution (A6, PEO, and ini-tiator in ethanol) is placed in a spinneret and a volt-age is applied. The polymer is collected either on agrounded metal sheet or a rotating shaft, exposed toultraviolet light for crosslinking, and then exposedto water for subsequent PEO removal.

SEM images of electrospun fibrous scaffolds at avariety of PEO:A6 ratios are shown in Figure 3(A).Distinct fibers are seen with morphological changesas the A6 content is increased. Specifically, the fiberdiameter increased and more welding is found atfiberfiber junctions with decreasing PEO content.Fiber diameter increased significantly for A6 con-tents greater than 20% [p < 0.05, Fig. 3(B)]. Fromthese initial SEM characterizations, the 65:35 PEO:A6ratio was selected based on its ability to maintainfiber form and limit junction formation, while limit-ing PEO content.

Reaction characterization

After formation, the crosslinking of the selected65:35 PEO:A6 scaffold with ultraviolet light was

monitored through the disappearance of the reactiveacrylate peak with time using ATR-FTIR [Fig. 4(A)].Through these measurements, the double bond con-version was quantified as a function of polymeriza-

Figure 1. A: Synthesis scheme and structure of macromerA6 used for electrospinning. B: Mass loss behavior for net-

works formed from A6 in slab form (n 5 3). Bovine mesen-chymal stem cell adhesion to films of networks formed frommacromer A6 (C) and control tissue culture polystyrene (D)after 7 days. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

Figure 2. Schematic of the electrospinning process. A so-lution of the macromer, PEO, and photoinitiator in 90%ethanol is placed in the spinneret to form a fibrous mat ofthe macromer and PEO. This mat is then exposed to ultra-violet light for crosslinking and then water for partial re-moval of PEO. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]




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tion time [Fig. 4(B)]. Maximal conversion was reachedafter 8 min and was high (>95%), indicating suc-cessful crosslinking. The structural integrity of thescaffold was assessed visually and by placing the scaf-fold in methylene chloride to dissolve away unreactedcomponents. SEM images showed no changes in fiberstructure with crosslinking, and failure of the samplesto dissolve in methylene chloride demonstrated uni-formity in sample integrity with depth.

Mechanical properties

Mechanical properties of formed scaffolds weredetermined before and after crosslinking using auniaxial tensile test to failure. Results from mechani-cal testing illustrated that crosslinked samples had a

higher tensile modulus than their uncrosslinkedcounterparts (p < 0.001). Specifically, for crosslinkedscaffolds, a modulus of 1.3, 23, and 12 MPa[Fig. 5(A)] was determined in nonaligned andaligned samples tested in the fiber direction, andaligned samples tested perpendicular to the fiberdirection samples, respectively. For comparison,uncrosslinked scaffolds had a modulus of 0.3, 6,and 1.1 MPa for these same conditions. Addition-ally, aligned scaffolds exhibited a mechanical anisot-

ropy ratio (parallel/perpendicular properties) of 5.45before crosslinking and 1.91 after crosslinking, indi-cating structural and mechanical differences depend-ent on directional orientation. Crosslinked, alignedfibrous scaffolds tested in the parallel direction alsoexhibited significantly higher tensile moduli com-pared to bulk A6 polymer slabs, crosslinked nona-

Figure 3. Morphology of electrospun fibers. A: Electrospun fibers of PEO and macromer A6 at various mass ratios(PEO:A6); scale bar 5 10 lm. B: Fiber diameter as a function of PEO concentration. *p < 0.05 compared to 100% PEO

fibers.

Figure 4. Photopolymerization of fibrous scaffolds. A: Decrease in the acrylate peak with exposure to light. B: Double bond conversion with time.

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ligned scaffolds, and crosslinked aligned scaffoldstested in the perpendicular direction (p < 0.001). It isexpected that the fibrous scaffolds would have ahigher moduli than the A6 slabs because they containa second polymer component (PEO), which can con-tribute to the polymer mechanics. Additionally, there

is potential for polymer chains to align within a fiberduring electrospinning, which could influencemechanics, but would not occur in the polymer slabs.With exposure to an aqueous environment for 1, 3,and 6 days, scaffolds exhibited significantly lowertensile moduli when compared with initial constructs[7.5, 6.6, 6.9 MPa, respectively; p < 0.001; Fig.5(B)] as PEO eluted from the fibrous structures.

Mass loss and NMR characterization

Sample degradation was further analyzed with

submersion in an aqueous environment and moni-toring of the mass loss and NMR analysis of degra-dation products. The mass loss observed for thecrosslinked samples after submersion in waterincluded a very rapid loss of mass within the first6 h, followed by a more gradual release for the next5 days, and then a plateau [Fig. 6(A)]. Long-term deg-radation analysis was not performed on these sam-ples. SEM analysis of the fibrous scaffolds after massloss indicates some swelling and welding of the fiber

junctions [Fig. 6(B)]. NMR analysis of the initial35:65 A6:PEO solution spectrum identified peaks at4.2 and 3.6 ppm, which can be attributed to A6 and

an overlap of A6 and PEO, respectively [Fig. 7(A)].These peaks were analyzed in the supernatants ofthe mass loss samples and a representative spectrumis shown in Figure 7(B). Both visual analysis(decrease in peak from A6 compared to peak fromPEO) and quantification (Fig. 7, inset) indicate that

the primary component being released from the fibrous scaffolds after submersion in water is PEO.For instance, there is more than seven times as muchPEO in the release solution after 6 h compared to A6than in the initial solutions prepared for electrospin-ning. This ratio decreases with continued exposure,as the scaffold approaches a plateau in this initialmass loss regime.

DISCUSSION

Current tissue engineering regenerative solutionsthat utilize electrospinning are limited to the proper-ties offered by previously electrospun polymers. Inthis study, we address the need to increase the num-

ber of polymers that can be electrospun by introduc-ing photocrosslinkable PBAE macromers from alibrary of polymers with a wide range of proper-ties.21 The objective of this work was to develop anelectrospinning process that could be used to formfibrous scaffolds from a wide range of photocros-slinkable precursors. Photocrosslinked networks arefinding application for the engineering of numeroustissues.24,25 Only minimal work has been performed

Figure 5. Mechanical properties of electrospun scaffolds. A: Modulus for A6 slab (without PEO) and both nonaligned(NA) and aligned (A) scaffolds before (pre) and after (post) crosslinking tested both parallel (PA) and perpendicular (PE)to fiber alignment (n 5 6). B: Scaffold modulus (A-PA, post) initially and after incubation in water for 1, 3, and 6 days (n5 6). *p < 0.05 compared to uncrosslinked scaffold of same orientation and **p < 0.05 compared to samples before incuba-tion in water.




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on the electrospinning of methacrylated or acrylatedprecursors and these have been primarily directedtoward the production of swollen hydrogels andcoatings,26,27 not stable scaffolds with sufficient me-chanical integrity for use in three-dimensional tissueengineering applications. For this initial study, weselected one macromer with potential properties ofinterest for tissue regeneration scaffolds. MacromerA6 was selected through a screening process that

illustrated that networks formed from the macromerexhibited a gradual hydrolytic degradation profileand enabled positive cellular interactions comparableto a control of TCPS. While the macromer A6 ispromising for tissue engineering applications, theobjective of this work was primarily to develop aprocedure for the electrospinning of radically poly-merizable macromers in general, and not to identifythis specific chemistry as optimal for tissue engineer-ing. In the future, this technology can be imple-mented to electrospin any candidate from the PBAElibrary at different molecular weights to tune fibrousscaffold properties for specific applications.

The first step in this process was the identificationof a method to electrospin low molecular weightmacromers. Typically, high molecular weight poly-mers are chosen because of their desired molecularinteractions and inherent viscosity during the spin-ning process. After macromer A6 failed to formfibers in initial experiments, it was coelectrospunalong with a high molecular weight polymer, PEO.Coelectrospinning of blended polymers has beenpreviously employed to exploit beneficial features ofthe individual components.28 In this study, PEO waschosen for this purpose as it is readily electrospun,will assist in fiber formation, will not interact with

Figure 7. NMR spectrum of the initial electrospun solu-tion of A6 and PEO (A) and a representative spectrum ofthe released products after 6 h (B). Inset: Normalized com-position of the released products compared to the initialelectrospun sample. These results indicate that the majorityof the mass loss initially is from released PEO and a smallamount of A6.

Figure 6. Mass loss for electrospun and crosslinked fibers. A: Mass loss with incubation in water for up to a week ( n 5 3per time point). B: SEM image of scaffold 4 days after incubation in water; scale bar 5 25 lm.

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the PBAE macromer during crosslinking, and willthen elute from the crosslinked network rapidlywith submersion in an aqueous environment. Themacromer was successfully electrospun and formedfibrous scaffolds, with morphological changes appa-rent in the scaffold depending on the ratio of the

photocrosslinkable macromer and carrier polymer.Specifically, the fiber diameter and junction weldingincreased with more macromer. The change in fiberdiameter could also be influenced by the viscosity ofthe electrospun solution which changes with theaddition of the PEO component.

One composition (65:35 PEO:A6) was chosen forsubsequent analysis based on morphological featuresand to minimize the PEO content. When exposed tolight, crosslinked networks formed, as evidenced bythe decrease in the acrylate peak, an increase in scaf-fold modulus, and a resistance to dissolution afterultraviolet light exposure. The reaction behavior ledto very high conversions, which indicates a low con-centration of unreacted macromers that can elutefrom the crosslinked structure. While not tested spe-cifically in this work, we speculate that the increasesin mechanical properties of the formed scaffolds afterexposure to ultraviolet light results from the forma-tion of both intra- and inter-strand covalent cross-links. Increases in interstrand physical linkages (or

bonding), via variations in solvent formulation, haspreviously been shown to increase the mechanicalproperties of scaffolds composed of segmented poly-urethane.29

The mechanical strength of both nonaligned andaligned scaffolds formed from this A6-PEO blendbefore and after covalent crosslinking, as well as themass loss profile of these constructs and the corre-sponding effect on mechanical properties wasdetermined. As reported by numerous other au-thors,6,7,9,30 deposition of electrospun polymers ontoa rotating mandrel produces physical alignment ofthe fibers with significant mechanical anisotropy. Inthis manuscript, we show for the first time the abil-ity of a member of the PBAE library to be spun inthis same manner.31 Interestingly, and unlike previ-ous reports,9,30 the aligned scaffolds were found to

have greater moduli than nonaligned scaffolds whentested in either the fiber or crossfiber directions. Themorphology of the fibers may explain this finding asthe aligned scaffolds appear to have an increasednumber of interfiber contacts (with fibers sometimestracking along one another for significant lengths). Ifinterfiber crosslinks form upon ultraviolet light expo-sure, this lengthy contact region would lead to morecrosslinking between fibers in the aligned scaffoldsover the nonaligned scaffolds with their more dis-persed fiber junctions. Additionally, this could leadto direction-dependent differences in mechanics pre-and post-crosslinking in aligned scaffolds, which was

observed with the decrease in mechanical anisotropywith ultraviolet light exposure. In this work, an 10-fold increase in modulus is seen in the perpendiculardirection with crosslinking, whereas only a 34-foldincrease is noted in the parallel direction, where PEOand fiber alignment already dominated the mechanics.

In our degradation studies, both mass loss andmechanical testing indicate that polymer is beingreleased from the fibrous scaffolds after immersionin water. 1H NMR analysis indicates that this is pri-marily the PEO component, with a small amount ofA6, which may be attributed to a small soluble frac-tion or short-term degradation. The rapid decreasein mechanics indicates that the carrier polymer pro-vides the scaffold with a significant portion of its ini-tial mechanical properties, and that the crosslinkedA6 macromer defines the properties after mass losshas plateaued. Also, the mass loss results (


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trolled composite scaffold properties. Thus, the abilityto tune these properties in individual polymer scaf-folds and optimally combine them using a widerange of photocrosslinkable polymers will be essen-tial in the next generation of functional tissue engi-neering of fibrous tissues.

CONCLUSIONS

In this study, fibrous scaffolds were successfullyelectrospun from a PBAE macromer with potentialapplication in tissue engineering using PEO as a car-rier polymer. When exposed to ultraviolet light inthe presence of a photoinitiator, crosslinked net-works were formed with almost 100% conversion ofthe double bonds and a corresponding increase inmechanical strength. Both nonaligned and aligned fi-

brous meshes can be formed from this process.When exposed to water, the scaffolds decrease inmass and mechanics, due primarily to release of thecarrier polymer. The ability to electrospin photocros-slinkable and low molecular weight macromers willallow for the production of fibrous scaffolds with awide range of degradation and mechanical proper-ties, particularly from the PBAE library. This willfurther expand the application of fibrous scaffolds toa wider range of tissue engineering approaches.

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andrea r. tan et al- electrospinning of photocrosslinked and degradable fibrous scaffolds

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