IOP PUBLISHING BIOMEDICAL MATERIALS

Biomed Mater 3 (2008) 034104 (8pp) doi1010881748-604133034104

Biodegradable and radically polymerizedelastomers with enhanced processingcapabilitiesJamie L Ifkovits1 Robert F Padera2 and Jason A Burdick1

1 Department of Bioengineering University of Pennsylvania Philadelphia PA 19104 USA2 Department of Pathology Brigham and Womenrsquos Hospital Harvard Medical School BostonMA 02115 USA

E-mail burdick2seasupennedu

Received 26 November 2007Accepted for publication 13 February 2008Published 8 August 2008Online at stacksioporgBMM3034104

AbstractThe development of biodegradable materials with elastomeric properties is beneficial for avariety of applications including for use in the engineering of soft tissues Although othershave developed biodegradable elastomers they are restricted by their processing at hightemperatures and under vacuum which limits their fabrication into complex scaffolds Toovercome this we have modified precursors to a tough biodegradable elastomer poly(glycerolsebacate) (PGS) with acrylates to impart control over the crosslinking process and allow formore processing options The acrylated-PGS (Acr-PGS) macromers are capable ofcrosslinking through free radical initiation mechanisms (eg redox and photo-initiatedpolymerizations) Alterations in the molecular weight and acrylation of the Acr-PGS led tochanges in formed network mechanical properties In general Youngrsquos modulus increasedwith acrylation and the strain at break increased with molecular weight when the acrylation was held constant Based on the mechanical properties one macromer was furtherinvestigated for in vitro and in vivo degradation and biocompatibility A mild to moderateinflammatory response typical of implantable biodegradable polymers was observed evenwhen formed as an injectable system with redox initiation Moreover fibrous scaffolds ofAcr-PGS and a carrier polymer poly(ethylene oxide) were prepared via an electrospinningand photopolymerization technique and the fiber morphology was dependent on the ratio ofthese components This system provides biodegradable polymers with tunable properties andenhanced processing capabilities towards the advancement of approaches in engineering softtissues

(Some figures in this article are in colour only in the electronic version)

1 Introduction

The well-known tissue engineering paradigm accounts forthe importance of scaffolds cells and growth factors andcombinations of these components for the successful designand integration of constructs into living systems to enhancetissue regeneration [1] It is generally believed that cells eitherdelivered or from surrounding tissues receive necessary cuesfrom their microenvironment which consists of both matrix(eg mechanics chemistry) and soluble factors [2 3] With

this in mind the chemical and physical properties of scaffoldsare of vital importance in controlling cellular behaviors (egdifferentiation matrix production) and in the overall successof the construct [2 4ndash9]

Scaffolds may comprise natural enzymatically degradablebiopolymers (eg hyaluronic acid) or synthetic polymers(eg polyurethanes) which are typically biodegradabledepending on the desired application and in vivo environment[10 11] One advantage to using synthetic polymersis the ability to tailor scaffold mechanical properties

1748-604108034104+08$3000 1 copy 2008 IOP Publishing Ltd Printed in the UK

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

and degradation kinetics through chemistry and processing[2 8 12] Synthetic materials frequently have moduli of theorder of MPa and GPa [8 9 11 13] whereas the elasticmodulus of many tissues is of the order of Pa and kPa [9 13]While hydrogels may have moduli of the order of many nativetissues [14] they are by definition hydrophilic materialswhich generally implies minimal protein adsorption andconsequently minimal cell attachment compared to theirhydrophobic polymeric counterparts [15] Furthermore manytissues exhibit elasticity such that they can function and recoverin the mechanically dynamic environment that exists in thebody [6 10 13 16] Therefore investigators have beenmotivated to synthesize and develop novel materials that bettermimic the stiffness and elasticity of native tissues [2 6 1013 16ndash18]

Wang and colleagues [16] synthesized a toughbiodegradable elastomer poly(glycerol sebacate) (PGS) thathas potential for the engineering of soft tissues due to itsmechanical properties and biocompatibility [13 16 19]However the curing of PGS requires high temperature andvacuum conditions [16 19] which makes processing intocomplex scaffolds difficult and in vivo crosslinking impossibleRecently Nijst et al [18] reported the modification offree hydroxy groups on the PGS prepolymer with acrylatefunctional groups to form an acrylated PGS (Acr-PGS)macromer The introduction of the acrylate functionalityintroduces control over the crosslinking and thus expandsupon the current processing options of this elastomer Wealso report here the modification of PGS precursors with theacrylate functionality but investigate the role of both molecularweight and degree of acrylate substitution on the formednetworks properties Since crosslinking of the vinylic bondson the Acr-PGS can occur via both redox and photo-initiatedfree radical polymerizations [20] we also explore thesedifferent mechanisms for enhanced processing of the networks(eg injectability and electrospinning) Additionally thesenetworks could be crosslinked using Michael-type additionreactions with the addition of a multifunctional nucleophile(eg di-thiol) but that approach is not shown here

Redox initiation is commonly used clinically topolymerize poly(methyl methacrylate) (PMMA)-based bonecements in vivo [21ndash23] Generally a bi-component systemsuch as that of benzoyl peroxide and NN-dimethyl-p-toluidine is used to promote initiation upon the mixingof the two components allowing for injectable applications[21ndash23] With this process it is possible for the minimallyinvasive delivery of injectable polymer formulations to remoteareas of the body where light penetration may not be possible[24] Furthermore injectable polymers can be formed directlyin a defect and good contact between the polymer andsurrounding tissue is possible [11 25] Therefore injectablepolymers are useful for a wide range of applications includingdrug delivery vehicles tissue adhesives and as tissue barriersand scaffolds [25ndash28]

Photoinitiated polymerizations are also useful for awide range of applications particularly due to the spatialand temporal control afforded during processing [11 29]Additionally polymerization exotherms and gelation times can

be controlled by simply varying the initiator concentration andlight intensity [11] These advantages of photopolymerizationmake it possible for the formation of complex scaffolds in both2D and 3D [30ndash32] We recently used photopolymerizationand electrospinning to generate fibrous scaffolds with bothisotropic and anisotropic structures from biodegradablepolymers [33] Electrospinning is a technique in which amat of continuous fibers is created by applying a voltageto a polymer solution [34 35] Fibrous scaffolds may beadvantageous for tissue engineering due to their high surface-to-mass ratio and their ability to mimic the native extracellularmatrix size and architecture [2 34] The ability to crosslinkthese elastomers using free radical polymerizations now opensup the possibility of processing into fibrous scaffolds

The overall objective of this work was to developradically polymerizable macromers that form biodegradableelastomeric networks upon crosslinking To accomplish thisa range of Acr-PGS macromers were synthesized and theirnetwork properties (eg degradation and mechanics) werecharacterized upon crosslinking The reaction behaviorsfor both redox and photo-initiated polymerizations and thetissue response to these biodegradable elastomers includinginjectable formulations were also investigated Finally theprocessing of the Acr-PGS macromers into fibrous scaffoldsvia electrospinning was explored

2 Experimental details

21 Macromer synthesis and characterization

All reagents were used as received from Sigma-Aldrich(St Louis MO) unless otherwise noted The prepolymer wasformed via the condensation reaction of equimolar amounts ofglycerol (ThermoFisher Scientific Waltham MA) and sebacicacid The reagents were combined and stirred at 120 C under anitrogen atmosphere for approximately 2 h and then a vacuumof 19 mbar was applied for various amounts of time (26ndash63 h) to obtain prepolymers of varying molecular weightsFor acrylation the prepolymer was dissolved in methylenechloride (ThermoFisher Scientific) containing triethylamine(TEA equimolar to acryloyl chloride) and 500 ppm4-methoxyphenol (inhibitor 10 wt in methylene chloride)Various molar ratios of acryloyl chloride (110 vvin methylene chloride) were dripped into the solution Thesevalues were calculated using the estimation that two of thethree hydroxy groups present in glycerol reacted with thesebacic acid and provide a range of overall acrylationsAn additional 500 ppm 4-methoxyphenol was added to thereaction chamber and a rotary evaporator (40 C 450 mbar)was used to remove the methylene chloride Ethyl acetatewas added to the reaction flask and the solution was vacuumfiltered to remove the TEA salts and washed three timeswith 10 mM hydrochloric acid (ThermoFisher Scientific)Ethyl acetate was removed via rotovapping (40 C 99 mbar)to leave a viscous liquid which was redissolved in methylenechloride and stored at 4 C The prepolymer and macromermolecular weights and chemical structures were verifiedusing GPC (Waters GPC System Milford MA) and 1H

2

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

NMR spectroscopy (Bruker Advance 360 MHz BrukerBillerica MA) and the results are outlined in figure 1

22 Reaction characterization

Macromers were mixed with 05 wt of the photoinitiator22-dimethoxy-2-phenylacetophenone (DMPA 10 wtmethylene chloride) and excess methylene chloride wasremoved by rotovapping The reaction behavior wasmonitored using real time attenuated total internal reflectionFourier transform infrared spectroscopy with a zinc seleniumcrystal (ATR-FTIR Nicolet 6700 Thermo Electron WalthamMA) A sample of the macromer with photoinitiator wasplaced directly on the surface of the crystal covered witha glass coverslip and monitored in real time with exposureto ultraviolet light (15 mW cmminus2 365 nm Omnicure S100EXFO Quebec) In the case of redox initiation sampleswere prepared by the addition of 10 wt benzoyl peroxide(BPO 10 wt in methylene chloride) or NN-dimethyl-p-toluidine (DMPT) to the macromermethylene chloridesolution Methylene chloride was removed by rotovappingand the samples were dissolved in 200 proof ethanol andtransferred to a dual barrel syringe (PlasPak Industries IncNorwich CT) which was subsequently placed into a 60 Coven overnight to remove any excess ethanol Sampleswere ejected from the dual barrel syringe directly onto thesurface of the crystal Reaction conversion was determinedby monitoring the change in the vinylic double bond peak(sim1635 cmminus1) however direct quantification was not possibledue to overlapping adjacent peaks and an unstable baselineThe gelation times and reaction exotherms were quantifiedusing a slowly stirring stirbar (60 rpm) in a vial post-mixingand a thermocouple temperature probe

23 Degradation and material property characterization

For sample fabrication the macromerinitiator solutions werepoured into a 50 times 15 times 1 mm teflon mold and placedin an oven at 60 C overnight The construct was thencovered with a glass slide and polymerized with exposure tosim10 mW cmminus2 365 nm ultraviolet light (Blak-Ray UltravioletProducts Upland CA) for 10 min Polymer disks (1 mm thick5 mm diameter) were punched from the resulting polymerslabs To monitor in vitro degradation samples were weighedsubmerged in 150 mM NaCl PBS and placed on an orbitalshaker at 37 C At each time point (2 4 and 8 weeks) samples(n = 3) were removed lyophilized (Freezone 45 LabconcoKansas City MO) for 24 h and weighed to determine massloss For mechanical testing strips (15 times 5 times 1 mm) were cutfrom the slabs and tensile testing was conducted on an Instron5848 mechanical tester (Norwood MA) with a 500 N load cellat a strain rate of 01 sminus1

24 In vivo tissue response

Animals were cared for according to a protocol approvedby the University of Pennsylvania Institute for Animal andUse Committee Photopolymerized polymer slabs wereprepared as described above Polymer discs (1 mm thick

5 mm diameter) were punched and submerged in ethanolEthanol was evaporated off and the discs were placed undera germicidal ultraviolet lamp for 30 min Redox initiatedmacromer solutions were loaded into a sterile dual barrelsyringe followed by exposure to the germicidal lamp for30 min Precrosslinked and preweighed discs (n = 4 pertime point 4 discs per animal) were implanted subcutaneouslyinto the dorsal pocket of male SpraguendashDawley rats Redoxinitiator loaded macromer solutions were also injected (n = 4)into the dorsal pocket of a male SpraguendashDawley rat Theanimals were sacrificed at various time points (2 4 and8 weeks) and the polymer samples and surrounding tissuewas collected and fixed with 10 formalin for 24 h Standardhemotoxylin and eosin (HampE) staining of paraffin embeddedsections was used to investigate the tissue response Additionalsamples were removed to monitor in vivo degradationbehavior All tissue was excised from the sample prior tolyophilization to obtain the sample dry weight

25 Electrospinning

An electrospun Acr-PGS scaffold was prepared by firstdissolving the photoinitiatormacromer in 90 ethanol(50 wt) To improve electrospinning potential themacromerethanol solution was combined with varyingpercentages of 10 poly(ethylene oxide) (PEO) (200 kDaPolysciences Warrington PA) in 90 ethanol Varioussolutions containing different ratios of the Acr-PGSethanoland PEOethanol were electrospun in a horizontal setup usinga flow rate of 25 mL hminus1 distance to collection plate of15 cm and a +15 kV applied voltage (ES30 Gamma HighVoltage Ormond Beach FL) The scaffolds were crosslinkedpost-electrospinning using an ultraviolet lamp (Blak-RayUltraviolet Products Upland CA) in a nitrogen atmosphereScaffolds were gold sputter coated and viewed using scanningelectron microscopy (Penn Regional Nanotech Facility JEOL6400 SEM Tokyo Japan)

3 Results and discussion

When designing a scaffold for tissue engineering thereare several design criteria (eg mechanics degradationbiocompatibility) to keep in mind Many believe that itis important to closely match the biomaterial mechanicalproperties with those of the surrounding native tissue to assistin the gradual transfer of stresses from implant to the newlyformed tissue [7 16] Specifically the elasticity of tissuesis often overlooked in material design yet biodegradableelastomers may fill that need Elastomers are generally definedas lightly crosslinked polymers that easily and quickly undergolarge reversible deformations with complete recovery [20]These important features of elastomers (eg PGS) makethem attractive materials to alleviate the compliance mismatchproblem that often exists with synthetic polymeric implantsparticularly in the dynamic environment of the human body[16]

3

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 1 Synthetic scheme (A) and representative 1H NMR spectra of Acr-PGS macromers (B) Peak letters correspond to those in themacromer structure above Unlabeled peaks correspond to protons from the initiator inhibitor or unreacted glycerol (peak d)

Table 1 Summary of Acr-PGS macromers synthesized andinvestigated

Macromer Mn (kDa) Mw (kDa) acrylation

1 201 406 8802 201 406 2173 201 406 904 340 702 2175 491 226 1796 533 235 96

31 Macromer synthesis and network characterization

Our approach is to fabricate tissue engineering scaffoldsusing a modified PGS prepolymer (ie Acr-PGS) that can becrosslinked under mild and physiologic conditions The firststep in the synthesis of the Acr-PGS macromer (figure 1(A))is the combination of trifunctional glycerol and difunctionalsebacic acid via a polycondensation reaction in a 11 molarratio for varying amounts of time (26ndash63 h) The startingreagents were chosen because they are naturally present inthe body and have been previously approved by the US Foodand Drug Administration for medical applications [16] TheMw of the prepolymer as defined by GPC increased withreaction time and ranged from sim406 kDa to 2346 kDaillustrating the tunability of molecular weight (table 1)As with most condensation reactions prepolymers werepolydisperse (201ndash460) and generally increased with reactiontime Multifunctional Acr-PGS macromers were prepared byreaction of the prepolymer with varying amounts of acryloylchloride These amounts were defined assuming that twoof the three hydroxy groups on the glycerol reacted with thesebacic acid during the condensation reaction and were chosen

to provide a range of acrylations The Acr-PGS acrylationswere determined using 1H NMR (table 1)

As seen in figure 1 peaks at sim13 16 and 23 ppmcorrespond to the protons in the olefin chain from sebacic acidand multiplets at sim42 and 52 ppm correspond to the protons inthe glycerol The peaks at sim59 61 and 63 ppm correspondto those of the functional acrylate group The acrylationwas determined by comparing the actual number of acrylategroups with the theoretical values for 100 incorporation ofthe acrylate group into the prepolymer The acrylationvalues range from sim96 to sim880 (table 1) These sixAcr-PGS macromers represent a range of molecular weightsand acrylations and thus provide insight into the relationshipsbetween macromer structure and network properties

The introduction of the acrylate functional groups wasalso confirmed using ATR-FTIR by visualization of thecharacteristic absorption of the acrylate group at sim1635 cmminus1

(figure 2) In general the intensity of this absorption increasedas the acrylation increased For example figure 2(A)displays the characteristic acrylate absorption for macromer 1and is representative of a high acrylation (880) whereasfigure 2(B) displays the characteristic acrylate absorption formacromer 6 and is representative of a low acrylation(96) A large difference in the acrylate absorption intensityis observed when comparing the two spectra

32 Network formation

The photopolymerization reaction was investigated byintroducing 05 wt photoinitiator (DMPA) into the Acr-PGSmacromer and monitoring the consumption of the acrylategroup peak in real time with exposure to 365 nm ultravioletlight (figure 2(A)) The maximum conversion occurred after

4

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 2 Consumption of the acrylate peak with time duringphotopolymerization of macromer 1 (05 wt DMPA light intensitysim15 mW cmminus2) (A) Consumption of the acrylate peak with timeduring the redox initiated polymerization of macromer 6 (10 wtBPO and DMPT) (B)

sim8 min The large difference in acrylate absorption betweeninitial and final time points and the near baseline level at thefinal time point indicate a high level of conversion of theacrylate group to crosslinks The redox-initiated crosslinkingwas also monitored by introduction of 10 wt of thebi-component BPO and DMPT initiation system which iscommonly used in bone cements [21ndash23] (figure 2(B)) Themaximum reaction conversion occurred after sim20 min Againthe difference in intensity between the initial and final timepoints indicates high conversion values

The gelation time was defined as the point when a slowlystirring stirbar was stopped after injection of the macromerinto a vial For this system gelation occurred at sim5 minand a maximum temperature of sim30 C (starting from roomtemperature) was observed The maximum conversion andgelation time can be tailored by altering the amount of initiatorincorporated into the macromer system as is the case withsimilar bi-component initiator systems [24 36] dependingon the application For example delivery of this materialto the heart through a catheter might require slower gelationthan direct injection into a defect The minimal increase intemperature is also important if this polymer is to be usedas an injectable formulation to prevent temperature-inducedtissue necrosis

(A)

(B)

Figure 3 Representative tensile stress vs elongation plots fornetworks formed from the Acr-PGS macromers with various MWsand acrylations (A) Youngrsquos modulus (black) and strain at break(white) for networks formed from the various synthesizedmacromers (B) Further information on the macromers can be foundin table 1

33 Network mechanical properties

Polymer slabs for mechanical and degradation analysis wereprepared using photopolymerization Typical tensile stressversus elongation relationships for networks formed fromAcr-PGS macromers are shown in figure 3(A) It is importantto note that many of the samples broke at the clamp andthus could lead to lower than actual values for the strain at break Youngrsquos modulus was determined from theslope of the linear portion of the plot (lt20 strain) andvaried (sim015ndash30 MPa) depending on the Acr-PGS macromer(figure 3(B)) The strain at break also varied (sim5ndash200)depending on the Acr-PGS macromer used for networkformation In general Youngrsquos modulus increased as thedegree of acrylation increased for a given molecular weightAs expected the strain at break increased as Youngrsquosmodulus and acrylation decreased for a given molecularweight Furthermore Youngrsquos modulus and strain atbreak increased with increasing molecular weight for similardegrees of acrylation As seen in figure 3(B) macromers 3and 6 have similar Youngrsquos moduli (sim150 kPa) but their elongation at break varied by almost an order of magnitudeBased on these results it important to note that not all ofthe macromers formed elastomeric networks Therefore

5

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

Figure 4 In vivo (black) and in vitro (white) degradation results at2 4 and 8 weeks for networks formed from macromer 6

macromer 6 was selected for the remaining studies since itsmechanical properties are the most elastomeric

Relationships between macromer structure and networkproperties can easily be drawn from these data For instanceincreased acrylation leads to an increase in the numberof crosslinks formed which is associated with an increasein the modulus of a resulting material and a decrease inthe ability to elongate before failure Additionally moreelastomeric-like features are obtained as the molecular weightof the prepolymer is increased This is a clear demonstrationthat small modifications during synthesis can lead to drasticdifferences in the properties of the resulting material Thusthis same backbone chemistry can be used to develop materialssuitable for a wide range of applications For example a moreelastic and softer material (eg Youngrsquos modulus sim150 kPa)may be more ideal for cardiac tissue engineering whereasa less elastic and a stiffer material (eg Youngrsquos modulussim30 MPa) may be more ideal for bone tissue engineeringAlthough not investigated here dynamic fatigue testing wouldbe necessary to illustrate the potential of these materials towithstand the dynamic in vivo environment (eg beating ofthe heart)

34 Network degradation and in vivo tissue response

The in vivo and in vitro mass loss of photopolymerized samplesreached a maximum of sim37 and sim33 respectively at8 weeks (figure 4) The in vivo mass loss was potentiallygreater because of its location in a more dynamic environmentwhere there is more fluid exchange to remove any degradationproducts from the implant region and due to the presenceof enzymes compared to the in vitro environment Alsothe sample preparation was slightly different due to thesterilization of the in vivo samples Based on these massloss data it is suspected that this material would be suitablefor a variety of tissue engineering applications Since redox-intiated samples could not be massed prior to in vivo injectionthe degradation profile could not be monitored However itis anticipated that it would be comparable to its photoinitiatedcounterpart if similar conversions are reached

After two weeks of implantation the host reaction tothe polymer discs comprised granulation tissue with new

(A)

(C )

(B)

(D)

Figure 5 HampE staining for in vivo tissue response to networksformed from macromer 6 at 2 weeks (A) 4 weeks (B) 4 weeks viainjectable redox initiation (C) and 8 weeks (D) (T tissue Ppolymer scale bar = 100 microm)

blood vessels loose connective tissue formation and mildchronic inflammation Macrophages and foreign body giantcells were present at the polymer-tissue interface (figure 5)At the 4 and 8 week time points a thin fibrous capsule ispresent around the implant with minimal associated chronicinflammation (figures 5(B) and (D) respectively) There isno evidence of inflammation or necrosis within the adjacentsubcutaneous fibroadipose tissue skin adnexal structures ordeep skeletal muscle This represents a typical host responseto a biocompatible material The reaction to the injectedpolymer (figure 5(C)) contained slightly more perivascularchronic inflammation in the surrounding host tissue butwithout evidence of necrosis or tissue damage This slightdifference in response to the injected polymer may be due toa mild toxicity associated with the initiators or differences inthe polymer configuration and surface area as compared to thephotoinitiated samples

35 Electrospinning into fibrous scaffolds

Electrospinning has gained much attention in recent yearsas a method for generating fibrous scaffolds [34 35]Fibrous scaffolds are thought to be advantageous since theyclosely mimic the architecture and size-scale of the nativeextracellular matrix [2 34] Many synthetic and naturalpolymers have been successfully electrospun to date [34 37]however the need to electrospin more polymers with a rangeof material properties (eg mechanics and degradation)especially those of elastomers still exists Due to the lowmolecular weight and high polydispersity of Acr-PGS it wasnecessary to modify techniques to electrospin the macromersTan and colleagues recently used photopolymerization and

6

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

(C)

Figure 6 SEM images of electrospun Acr-PGSPEO scaffolds atratios of 3070 (A) 4060 (B) and 5050 (C) Scale bar = 50 microm

electrospinning to create isotropic and anisotropic scaffoldsfrom low molecular weight biodegradable macromers byusing 200 kDa PEO as a carrier polymer [33] Thesame processing protocol was followed to prepare mats ofelectrospun and photocrosslinkable Acr-PGSPEO containinga photoinitiator Mats were electrospun at various ratios ofAcr-PGS to PEO solutions and crosslinked with ultravioletlight prior to visualization under SEM (figure 6) A ratio of3070 PGSPEO (figure 6(A)) produced a mat with the bestmechanical integrity and most distinct fibers which is lostwhen the PEO concentration is too low (figures 6(B) and (C))At this point a thorough characterization of mechanics andcellular interactions has not been performed on these scaffoldsbut our ability to obtain the proper structures motivates furtherexploration of these materials

4 Conclusions

In this study radically polymerized networks with tunablemechanical properties were successfully synthesized andcharacterized Notably an increase in Youngrsquos modulus withincreasing acrylation as well as an increase in the strainat break with increasing molecular weight were observedindicating that these properties can be tuned through the designof the macromer and that networks with elastomeric propertiescan be obtained The reaction behavior was rapid andreached high conversions with both redox and photoinitiatedpolymerizations The networks also degraded more rapidlyin vivo and only mild inflammation was seen even withinjectable formulations Moreover this polymer system couldbe processed into fibrous scaffolds using electrospinning andPEO as a carrier polymer This biodegradable and elastomericpolymer system can be further explored for the engineering

of numerous tissues where elasticity is an importantparameter

Acknowledgments

The authors would like to acknowledge Joshua S Katz forhelpful synthetic discussions and Cindy Chung for assistancewith animal surgeries This work was funded by the AmericanChemical Society Petroleum Research Fund a pilot grant fromthe National Science Foundation Materials Research Scienceand Engineering Center at the University of Pennsylvania andan Ashton Fellowship to JLI

References

[1] Langer R and Vacanti J P 1993 Tissue engineering Science260 920ndash6

[2] Lavik E and Langer R 2004 Tissue engineering current stateand perspectives Appl Microbiol Biotechnol 65 1ndash8

[3] Nerem R M 2006 Tissue engineering the hope the hype andthe future Tissue Eng 12 1143ndash50

[4] Brey D M Ifkovits J L Mozia R I Katz J S and Burdick J A2008 Controlling poly(β-amino ester) network propertiesthrough macromer branching Acta Biomater 4 207ndash17

[5] Chung C Mesa J Randolph M A Yaremchuk M andBurdick J A 2006 Influence of gel properties onneocartilage formation by auricular chondrocytesphotoencapsulated in hyaluronic acid networks J BiomedMater Res A 77 518ndash525

[6] Yang J Webb A R and Ameer G A 2004 Novel citricacid-based biodegradable elastomers for tissue engineeringAdv Mater 16 511

[7] Guan J Stankus J J and Wagner W R 2007 Biodegradableelastomeric scaffolds with basic fibroblast growth factorrelease J Control Release 120 70ndash8

[8] Gunatillake P A and Adhikari R 2003 Biodegradable syntheticpolymers for tissue engineering Eur Cells Mater 5 1ndash16discussion 16

[9] Levental I Georges P C and Janmey P A 2007 Soft biologicalmaterials and their impact on cell function Soft Matter3 299ndash306

[10] Guan J J Sacks M S Beckman E J and Wagner W R 2002Synthesis characterization and cytocompatibility ofefastomeric biodegradable poly(ester-urethane)ureas basedon poly(caprolactone) and putrescine J Biomed MaterRes 61 493ndash503

[11] Ifkovits J L and Burdick J A 2007 Reviewphotopolymerizable and degradable biomaterials for tissueengineering applications Tissue Eng 13 2369ndash85

[12] Anderson D G et al 2006 A combinatorial library ofphotocrosslinkable and degradable materials Adv Mater18 2614

[13] Webb A R Yang J and Ameer G A 2004 Biodegradablepolyester elastomers in tissue engineering Expert OpinBiol Ther 4 801ndash12

[14] Temenoff J S Athanasiou K A LeBaron R G and Mikos A G2002 Effect of poly(ethylene glycol) molecular weight ontensile and swelling properties of oligo(poly(ethyleneglycol) fumarate) hydrogels for cartilage tissue engineeringJ Biomed Mater Res 59 429ndash37

[15] Nuttelman C R Mortisen D J Henry S M and Anseth K S2001 Attachment of fibronectin to poly(vinyl alcohol)hydrogels promotes NIH3T3 cell adhesion proliferationand migration J Biomed Mater Res 57 217ndash23

[16] Wang Y D Ameer G A Sheppard B J and Langer R 2002A tough biodegradable elastomer Nat Biotechnol20 602ndash6

7

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

NMR spectroscopy (Bruker Advance 360 MHz BrukerBillerica MA) and the results are outlined in figure 1

22 Reaction characterization

Macromers were mixed with 05 wt of the photoinitiator22-dimethoxy-2-phenylacetophenone (DMPA 10 wtmethylene chloride) and excess methylene chloride wasremoved by rotovapping The reaction behavior wasmonitored using real time attenuated total internal reflectionFourier transform infrared spectroscopy with a zinc seleniumcrystal (ATR-FTIR Nicolet 6700 Thermo Electron WalthamMA) A sample of the macromer with photoinitiator wasplaced directly on the surface of the crystal covered witha glass coverslip and monitored in real time with exposureto ultraviolet light (15 mW cmminus2 365 nm Omnicure S100EXFO Quebec) In the case of redox initiation sampleswere prepared by the addition of 10 wt benzoyl peroxide(BPO 10 wt in methylene chloride) or NN-dimethyl-p-toluidine (DMPT) to the macromermethylene chloridesolution Methylene chloride was removed by rotovappingand the samples were dissolved in 200 proof ethanol andtransferred to a dual barrel syringe (PlasPak Industries IncNorwich CT) which was subsequently placed into a 60 Coven overnight to remove any excess ethanol Sampleswere ejected from the dual barrel syringe directly onto thesurface of the crystal Reaction conversion was determinedby monitoring the change in the vinylic double bond peak(sim1635 cmminus1) however direct quantification was not possibledue to overlapping adjacent peaks and an unstable baselineThe gelation times and reaction exotherms were quantifiedusing a slowly stirring stirbar (60 rpm) in a vial post-mixingand a thermocouple temperature probe

23 Degradation and material property characterization

For sample fabrication the macromerinitiator solutions werepoured into a 50 times 15 times 1 mm teflon mold and placedin an oven at 60 C overnight The construct was thencovered with a glass slide and polymerized with exposure tosim10 mW cmminus2 365 nm ultraviolet light (Blak-Ray UltravioletProducts Upland CA) for 10 min Polymer disks (1 mm thick5 mm diameter) were punched from the resulting polymerslabs To monitor in vitro degradation samples were weighedsubmerged in 150 mM NaCl PBS and placed on an orbitalshaker at 37 C At each time point (2 4 and 8 weeks) samples(n = 3) were removed lyophilized (Freezone 45 LabconcoKansas City MO) for 24 h and weighed to determine massloss For mechanical testing strips (15 times 5 times 1 mm) were cutfrom the slabs and tensile testing was conducted on an Instron5848 mechanical tester (Norwood MA) with a 500 N load cellat a strain rate of 01 sminus1

24 In vivo tissue response

Animals were cared for according to a protocol approvedby the University of Pennsylvania Institute for Animal andUse Committee Photopolymerized polymer slabs wereprepared as described above Polymer discs (1 mm thick

5 mm diameter) were punched and submerged in ethanolEthanol was evaporated off and the discs were placed undera germicidal ultraviolet lamp for 30 min Redox initiatedmacromer solutions were loaded into a sterile dual barrelsyringe followed by exposure to the germicidal lamp for30 min Precrosslinked and preweighed discs (n = 4 pertime point 4 discs per animal) were implanted subcutaneouslyinto the dorsal pocket of male SpraguendashDawley rats Redoxinitiator loaded macromer solutions were also injected (n = 4)into the dorsal pocket of a male SpraguendashDawley rat Theanimals were sacrificed at various time points (2 4 and8 weeks) and the polymer samples and surrounding tissuewas collected and fixed with 10 formalin for 24 h Standardhemotoxylin and eosin (HampE) staining of paraffin embeddedsections was used to investigate the tissue response Additionalsamples were removed to monitor in vivo degradationbehavior All tissue was excised from the sample prior tolyophilization to obtain the sample dry weight

25 Electrospinning

An electrospun Acr-PGS scaffold was prepared by firstdissolving the photoinitiatormacromer in 90 ethanol(50 wt) To improve electrospinning potential themacromerethanol solution was combined with varyingpercentages of 10 poly(ethylene oxide) (PEO) (200 kDaPolysciences Warrington PA) in 90 ethanol Varioussolutions containing different ratios of the Acr-PGSethanoland PEOethanol were electrospun in a horizontal setup usinga flow rate of 25 mL hminus1 distance to collection plate of15 cm and a +15 kV applied voltage (ES30 Gamma HighVoltage Ormond Beach FL) The scaffolds were crosslinkedpost-electrospinning using an ultraviolet lamp (Blak-RayUltraviolet Products Upland CA) in a nitrogen atmosphereScaffolds were gold sputter coated and viewed using scanningelectron microscopy (Penn Regional Nanotech Facility JEOL6400 SEM Tokyo Japan)

3 Results and discussion

When designing a scaffold for tissue engineering thereare several design criteria (eg mechanics degradationbiocompatibility) to keep in mind Many believe that itis important to closely match the biomaterial mechanicalproperties with those of the surrounding native tissue to assistin the gradual transfer of stresses from implant to the newlyformed tissue [7 16] Specifically the elasticity of tissuesis often overlooked in material design yet biodegradableelastomers may fill that need Elastomers are generally definedas lightly crosslinked polymers that easily and quickly undergolarge reversible deformations with complete recovery [20]These important features of elastomers (eg PGS) makethem attractive materials to alleviate the compliance mismatchproblem that often exists with synthetic polymeric implantsparticularly in the dynamic environment of the human body[16]

3

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 1 Synthetic scheme (A) and representative 1H NMR spectra of Acr-PGS macromers (B) Peak letters correspond to those in themacromer structure above Unlabeled peaks correspond to protons from the initiator inhibitor or unreacted glycerol (peak d)

Table 1 Summary of Acr-PGS macromers synthesized andinvestigated

Macromer Mn (kDa) Mw (kDa) acrylation

1 201 406 8802 201 406 2173 201 406 904 340 702 2175 491 226 1796 533 235 96

31 Macromer synthesis and network characterization

Our approach is to fabricate tissue engineering scaffoldsusing a modified PGS prepolymer (ie Acr-PGS) that can becrosslinked under mild and physiologic conditions The firststep in the synthesis of the Acr-PGS macromer (figure 1(A))is the combination of trifunctional glycerol and difunctionalsebacic acid via a polycondensation reaction in a 11 molarratio for varying amounts of time (26ndash63 h) The startingreagents were chosen because they are naturally present inthe body and have been previously approved by the US Foodand Drug Administration for medical applications [16] TheMw of the prepolymer as defined by GPC increased withreaction time and ranged from sim406 kDa to 2346 kDaillustrating the tunability of molecular weight (table 1)As with most condensation reactions prepolymers werepolydisperse (201ndash460) and generally increased with reactiontime Multifunctional Acr-PGS macromers were prepared byreaction of the prepolymer with varying amounts of acryloylchloride These amounts were defined assuming that twoof the three hydroxy groups on the glycerol reacted with thesebacic acid during the condensation reaction and were chosen

to provide a range of acrylations The Acr-PGS acrylationswere determined using 1H NMR (table 1)

As seen in figure 1 peaks at sim13 16 and 23 ppmcorrespond to the protons in the olefin chain from sebacic acidand multiplets at sim42 and 52 ppm correspond to the protons inthe glycerol The peaks at sim59 61 and 63 ppm correspondto those of the functional acrylate group The acrylationwas determined by comparing the actual number of acrylategroups with the theoretical values for 100 incorporation ofthe acrylate group into the prepolymer The acrylationvalues range from sim96 to sim880 (table 1) These sixAcr-PGS macromers represent a range of molecular weightsand acrylations and thus provide insight into the relationshipsbetween macromer structure and network properties

The introduction of the acrylate functional groups wasalso confirmed using ATR-FTIR by visualization of thecharacteristic absorption of the acrylate group at sim1635 cmminus1

(figure 2) In general the intensity of this absorption increasedas the acrylation increased For example figure 2(A)displays the characteristic acrylate absorption for macromer 1and is representative of a high acrylation (880) whereasfigure 2(B) displays the characteristic acrylate absorption formacromer 6 and is representative of a low acrylation(96) A large difference in the acrylate absorption intensityis observed when comparing the two spectra

32 Network formation

The photopolymerization reaction was investigated byintroducing 05 wt photoinitiator (DMPA) into the Acr-PGSmacromer and monitoring the consumption of the acrylategroup peak in real time with exposure to 365 nm ultravioletlight (figure 2(A)) The maximum conversion occurred after

4

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 2 Consumption of the acrylate peak with time duringphotopolymerization of macromer 1 (05 wt DMPA light intensitysim15 mW cmminus2) (A) Consumption of the acrylate peak with timeduring the redox initiated polymerization of macromer 6 (10 wtBPO and DMPT) (B)

sim8 min The large difference in acrylate absorption betweeninitial and final time points and the near baseline level at thefinal time point indicate a high level of conversion of theacrylate group to crosslinks The redox-initiated crosslinkingwas also monitored by introduction of 10 wt of thebi-component BPO and DMPT initiation system which iscommonly used in bone cements [21ndash23] (figure 2(B)) Themaximum reaction conversion occurred after sim20 min Againthe difference in intensity between the initial and final timepoints indicates high conversion values

The gelation time was defined as the point when a slowlystirring stirbar was stopped after injection of the macromerinto a vial For this system gelation occurred at sim5 minand a maximum temperature of sim30 C (starting from roomtemperature) was observed The maximum conversion andgelation time can be tailored by altering the amount of initiatorincorporated into the macromer system as is the case withsimilar bi-component initiator systems [24 36] dependingon the application For example delivery of this materialto the heart through a catheter might require slower gelationthan direct injection into a defect The minimal increase intemperature is also important if this polymer is to be usedas an injectable formulation to prevent temperature-inducedtissue necrosis

(A)

(B)

Figure 3 Representative tensile stress vs elongation plots fornetworks formed from the Acr-PGS macromers with various MWsand acrylations (A) Youngrsquos modulus (black) and strain at break(white) for networks formed from the various synthesizedmacromers (B) Further information on the macromers can be foundin table 1

33 Network mechanical properties

Polymer slabs for mechanical and degradation analysis wereprepared using photopolymerization Typical tensile stressversus elongation relationships for networks formed fromAcr-PGS macromers are shown in figure 3(A) It is importantto note that many of the samples broke at the clamp andthus could lead to lower than actual values for the strain at break Youngrsquos modulus was determined from theslope of the linear portion of the plot (lt20 strain) andvaried (sim015ndash30 MPa) depending on the Acr-PGS macromer(figure 3(B)) The strain at break also varied (sim5ndash200)depending on the Acr-PGS macromer used for networkformation In general Youngrsquos modulus increased as thedegree of acrylation increased for a given molecular weightAs expected the strain at break increased as Youngrsquosmodulus and acrylation decreased for a given molecularweight Furthermore Youngrsquos modulus and strain atbreak increased with increasing molecular weight for similardegrees of acrylation As seen in figure 3(B) macromers 3and 6 have similar Youngrsquos moduli (sim150 kPa) but their elongation at break varied by almost an order of magnitudeBased on these results it important to note that not all ofthe macromers formed elastomeric networks Therefore

5

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

Figure 4 In vivo (black) and in vitro (white) degradation results at2 4 and 8 weeks for networks formed from macromer 6

macromer 6 was selected for the remaining studies since itsmechanical properties are the most elastomeric

Relationships between macromer structure and networkproperties can easily be drawn from these data For instanceincreased acrylation leads to an increase in the numberof crosslinks formed which is associated with an increasein the modulus of a resulting material and a decrease inthe ability to elongate before failure Additionally moreelastomeric-like features are obtained as the molecular weightof the prepolymer is increased This is a clear demonstrationthat small modifications during synthesis can lead to drasticdifferences in the properties of the resulting material Thusthis same backbone chemistry can be used to develop materialssuitable for a wide range of applications For example a moreelastic and softer material (eg Youngrsquos modulus sim150 kPa)may be more ideal for cardiac tissue engineering whereasa less elastic and a stiffer material (eg Youngrsquos modulussim30 MPa) may be more ideal for bone tissue engineeringAlthough not investigated here dynamic fatigue testing wouldbe necessary to illustrate the potential of these materials towithstand the dynamic in vivo environment (eg beating ofthe heart)

34 Network degradation and in vivo tissue response

The in vivo and in vitro mass loss of photopolymerized samplesreached a maximum of sim37 and sim33 respectively at8 weeks (figure 4) The in vivo mass loss was potentiallygreater because of its location in a more dynamic environmentwhere there is more fluid exchange to remove any degradationproducts from the implant region and due to the presenceof enzymes compared to the in vitro environment Alsothe sample preparation was slightly different due to thesterilization of the in vivo samples Based on these massloss data it is suspected that this material would be suitablefor a variety of tissue engineering applications Since redox-intiated samples could not be massed prior to in vivo injectionthe degradation profile could not be monitored However itis anticipated that it would be comparable to its photoinitiatedcounterpart if similar conversions are reached

After two weeks of implantation the host reaction tothe polymer discs comprised granulation tissue with new

(A)

(C )

(B)

(D)

Figure 5 HampE staining for in vivo tissue response to networksformed from macromer 6 at 2 weeks (A) 4 weeks (B) 4 weeks viainjectable redox initiation (C) and 8 weeks (D) (T tissue Ppolymer scale bar = 100 microm)

blood vessels loose connective tissue formation and mildchronic inflammation Macrophages and foreign body giantcells were present at the polymer-tissue interface (figure 5)At the 4 and 8 week time points a thin fibrous capsule ispresent around the implant with minimal associated chronicinflammation (figures 5(B) and (D) respectively) There isno evidence of inflammation or necrosis within the adjacentsubcutaneous fibroadipose tissue skin adnexal structures ordeep skeletal muscle This represents a typical host responseto a biocompatible material The reaction to the injectedpolymer (figure 5(C)) contained slightly more perivascularchronic inflammation in the surrounding host tissue butwithout evidence of necrosis or tissue damage This slightdifference in response to the injected polymer may be due toa mild toxicity associated with the initiators or differences inthe polymer configuration and surface area as compared to thephotoinitiated samples

35 Electrospinning into fibrous scaffolds

Electrospinning has gained much attention in recent yearsas a method for generating fibrous scaffolds [34 35]Fibrous scaffolds are thought to be advantageous since theyclosely mimic the architecture and size-scale of the nativeextracellular matrix [2 34] Many synthetic and naturalpolymers have been successfully electrospun to date [34 37]however the need to electrospin more polymers with a rangeof material properties (eg mechanics and degradation)especially those of elastomers still exists Due to the lowmolecular weight and high polydispersity of Acr-PGS it wasnecessary to modify techniques to electrospin the macromersTan and colleagues recently used photopolymerization and

6

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

(C)

Figure 6 SEM images of electrospun Acr-PGSPEO scaffolds atratios of 3070 (A) 4060 (B) and 5050 (C) Scale bar = 50 microm

electrospinning to create isotropic and anisotropic scaffoldsfrom low molecular weight biodegradable macromers byusing 200 kDa PEO as a carrier polymer [33] Thesame processing protocol was followed to prepare mats ofelectrospun and photocrosslinkable Acr-PGSPEO containinga photoinitiator Mats were electrospun at various ratios ofAcr-PGS to PEO solutions and crosslinked with ultravioletlight prior to visualization under SEM (figure 6) A ratio of3070 PGSPEO (figure 6(A)) produced a mat with the bestmechanical integrity and most distinct fibers which is lostwhen the PEO concentration is too low (figures 6(B) and (C))At this point a thorough characterization of mechanics andcellular interactions has not been performed on these scaffoldsbut our ability to obtain the proper structures motivates furtherexploration of these materials

4 Conclusions

In this study radically polymerized networks with tunablemechanical properties were successfully synthesized andcharacterized Notably an increase in Youngrsquos modulus withincreasing acrylation as well as an increase in the strainat break with increasing molecular weight were observedindicating that these properties can be tuned through the designof the macromer and that networks with elastomeric propertiescan be obtained The reaction behavior was rapid andreached high conversions with both redox and photoinitiatedpolymerizations The networks also degraded more rapidlyin vivo and only mild inflammation was seen even withinjectable formulations Moreover this polymer system couldbe processed into fibrous scaffolds using electrospinning andPEO as a carrier polymer This biodegradable and elastomericpolymer system can be further explored for the engineering

of numerous tissues where elasticity is an importantparameter

Acknowledgments

The authors would like to acknowledge Joshua S Katz forhelpful synthetic discussions and Cindy Chung for assistancewith animal surgeries This work was funded by the AmericanChemical Society Petroleum Research Fund a pilot grant fromthe National Science Foundation Materials Research Scienceand Engineering Center at the University of Pennsylvania andan Ashton Fellowship to JLI

References

[1] Langer R and Vacanti J P 1993 Tissue engineering Science260 920ndash6

[2] Lavik E and Langer R 2004 Tissue engineering current stateand perspectives Appl Microbiol Biotechnol 65 1ndash8

[3] Nerem R M 2006 Tissue engineering the hope the hype andthe future Tissue Eng 12 1143ndash50

[4] Brey D M Ifkovits J L Mozia R I Katz J S and Burdick J A2008 Controlling poly(β-amino ester) network propertiesthrough macromer branching Acta Biomater 4 207ndash17

[5] Chung C Mesa J Randolph M A Yaremchuk M andBurdick J A 2006 Influence of gel properties onneocartilage formation by auricular chondrocytesphotoencapsulated in hyaluronic acid networks J BiomedMater Res A 77 518ndash525

[6] Yang J Webb A R and Ameer G A 2004 Novel citricacid-based biodegradable elastomers for tissue engineeringAdv Mater 16 511

[7] Guan J Stankus J J and Wagner W R 2007 Biodegradableelastomeric scaffolds with basic fibroblast growth factorrelease J Control Release 120 70ndash8

[8] Gunatillake P A and Adhikari R 2003 Biodegradable syntheticpolymers for tissue engineering Eur Cells Mater 5 1ndash16discussion 16

[9] Levental I Georges P C and Janmey P A 2007 Soft biologicalmaterials and their impact on cell function Soft Matter3 299ndash306

[10] Guan J J Sacks M S Beckman E J and Wagner W R 2002Synthesis characterization and cytocompatibility ofefastomeric biodegradable poly(ester-urethane)ureas basedon poly(caprolactone) and putrescine J Biomed MaterRes 61 493ndash503

[11] Ifkovits J L and Burdick J A 2007 Reviewphotopolymerizable and degradable biomaterials for tissueengineering applications Tissue Eng 13 2369ndash85

[12] Anderson D G et al 2006 A combinatorial library ofphotocrosslinkable and degradable materials Adv Mater18 2614

[13] Webb A R Yang J and Ameer G A 2004 Biodegradablepolyester elastomers in tissue engineering Expert OpinBiol Ther 4 801ndash12

[14] Temenoff J S Athanasiou K A LeBaron R G and Mikos A G2002 Effect of poly(ethylene glycol) molecular weight ontensile and swelling properties of oligo(poly(ethyleneglycol) fumarate) hydrogels for cartilage tissue engineeringJ Biomed Mater Res 59 429ndash37

[15] Nuttelman C R Mortisen D J Henry S M and Anseth K S2001 Attachment of fibronectin to poly(vinyl alcohol)hydrogels promotes NIH3T3 cell adhesion proliferationand migration J Biomed Mater Res 57 217ndash23

[16] Wang Y D Ameer G A Sheppard B J and Langer R 2002A tough biodegradable elastomer Nat Biotechnol20 602ndash6

7

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 1 Synthetic scheme (A) and representative 1H NMR spectra of Acr-PGS macromers (B) Peak letters correspond to those in themacromer structure above Unlabeled peaks correspond to protons from the initiator inhibitor or unreacted glycerol (peak d)

Table 1 Summary of Acr-PGS macromers synthesized andinvestigated

Macromer Mn (kDa) Mw (kDa) acrylation

1 201 406 8802 201 406 2173 201 406 904 340 702 2175 491 226 1796 533 235 96

31 Macromer synthesis and network characterization

Our approach is to fabricate tissue engineering scaffoldsusing a modified PGS prepolymer (ie Acr-PGS) that can becrosslinked under mild and physiologic conditions The firststep in the synthesis of the Acr-PGS macromer (figure 1(A))is the combination of trifunctional glycerol and difunctionalsebacic acid via a polycondensation reaction in a 11 molarratio for varying amounts of time (26ndash63 h) The startingreagents were chosen because they are naturally present inthe body and have been previously approved by the US Foodand Drug Administration for medical applications [16] TheMw of the prepolymer as defined by GPC increased withreaction time and ranged from sim406 kDa to 2346 kDaillustrating the tunability of molecular weight (table 1)As with most condensation reactions prepolymers werepolydisperse (201ndash460) and generally increased with reactiontime Multifunctional Acr-PGS macromers were prepared byreaction of the prepolymer with varying amounts of acryloylchloride These amounts were defined assuming that twoof the three hydroxy groups on the glycerol reacted with thesebacic acid during the condensation reaction and were chosen

to provide a range of acrylations The Acr-PGS acrylationswere determined using 1H NMR (table 1)

As seen in figure 1 peaks at sim13 16 and 23 ppmcorrespond to the protons in the olefin chain from sebacic acidand multiplets at sim42 and 52 ppm correspond to the protons inthe glycerol The peaks at sim59 61 and 63 ppm correspondto those of the functional acrylate group The acrylationwas determined by comparing the actual number of acrylategroups with the theoretical values for 100 incorporation ofthe acrylate group into the prepolymer The acrylationvalues range from sim96 to sim880 (table 1) These sixAcr-PGS macromers represent a range of molecular weightsand acrylations and thus provide insight into the relationshipsbetween macromer structure and network properties

The introduction of the acrylate functional groups wasalso confirmed using ATR-FTIR by visualization of thecharacteristic absorption of the acrylate group at sim1635 cmminus1

(figure 2) In general the intensity of this absorption increasedas the acrylation increased For example figure 2(A)displays the characteristic acrylate absorption for macromer 1and is representative of a high acrylation (880) whereasfigure 2(B) displays the characteristic acrylate absorption formacromer 6 and is representative of a low acrylation(96) A large difference in the acrylate absorption intensityis observed when comparing the two spectra

32 Network formation

The photopolymerization reaction was investigated byintroducing 05 wt photoinitiator (DMPA) into the Acr-PGSmacromer and monitoring the consumption of the acrylategroup peak in real time with exposure to 365 nm ultravioletlight (figure 2(A)) The maximum conversion occurred after

4

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 2 Consumption of the acrylate peak with time duringphotopolymerization of macromer 1 (05 wt DMPA light intensitysim15 mW cmminus2) (A) Consumption of the acrylate peak with timeduring the redox initiated polymerization of macromer 6 (10 wtBPO and DMPT) (B)

sim8 min The large difference in acrylate absorption betweeninitial and final time points and the near baseline level at thefinal time point indicate a high level of conversion of theacrylate group to crosslinks The redox-initiated crosslinkingwas also monitored by introduction of 10 wt of thebi-component BPO and DMPT initiation system which iscommonly used in bone cements [21ndash23] (figure 2(B)) Themaximum reaction conversion occurred after sim20 min Againthe difference in intensity between the initial and final timepoints indicates high conversion values

The gelation time was defined as the point when a slowlystirring stirbar was stopped after injection of the macromerinto a vial For this system gelation occurred at sim5 minand a maximum temperature of sim30 C (starting from roomtemperature) was observed The maximum conversion andgelation time can be tailored by altering the amount of initiatorincorporated into the macromer system as is the case withsimilar bi-component initiator systems [24 36] dependingon the application For example delivery of this materialto the heart through a catheter might require slower gelationthan direct injection into a defect The minimal increase intemperature is also important if this polymer is to be usedas an injectable formulation to prevent temperature-inducedtissue necrosis

(A)

(B)

Figure 3 Representative tensile stress vs elongation plots fornetworks formed from the Acr-PGS macromers with various MWsand acrylations (A) Youngrsquos modulus (black) and strain at break(white) for networks formed from the various synthesizedmacromers (B) Further information on the macromers can be foundin table 1

33 Network mechanical properties

Polymer slabs for mechanical and degradation analysis wereprepared using photopolymerization Typical tensile stressversus elongation relationships for networks formed fromAcr-PGS macromers are shown in figure 3(A) It is importantto note that many of the samples broke at the clamp andthus could lead to lower than actual values for the strain at break Youngrsquos modulus was determined from theslope of the linear portion of the plot (lt20 strain) andvaried (sim015ndash30 MPa) depending on the Acr-PGS macromer(figure 3(B)) The strain at break also varied (sim5ndash200)depending on the Acr-PGS macromer used for networkformation In general Youngrsquos modulus increased as thedegree of acrylation increased for a given molecular weightAs expected the strain at break increased as Youngrsquosmodulus and acrylation decreased for a given molecularweight Furthermore Youngrsquos modulus and strain atbreak increased with increasing molecular weight for similardegrees of acrylation As seen in figure 3(B) macromers 3and 6 have similar Youngrsquos moduli (sim150 kPa) but their elongation at break varied by almost an order of magnitudeBased on these results it important to note that not all ofthe macromers formed elastomeric networks Therefore

5

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

Figure 4 In vivo (black) and in vitro (white) degradation results at2 4 and 8 weeks for networks formed from macromer 6

macromer 6 was selected for the remaining studies since itsmechanical properties are the most elastomeric

Relationships between macromer structure and networkproperties can easily be drawn from these data For instanceincreased acrylation leads to an increase in the numberof crosslinks formed which is associated with an increasein the modulus of a resulting material and a decrease inthe ability to elongate before failure Additionally moreelastomeric-like features are obtained as the molecular weightof the prepolymer is increased This is a clear demonstrationthat small modifications during synthesis can lead to drasticdifferences in the properties of the resulting material Thusthis same backbone chemistry can be used to develop materialssuitable for a wide range of applications For example a moreelastic and softer material (eg Youngrsquos modulus sim150 kPa)may be more ideal for cardiac tissue engineering whereasa less elastic and a stiffer material (eg Youngrsquos modulussim30 MPa) may be more ideal for bone tissue engineeringAlthough not investigated here dynamic fatigue testing wouldbe necessary to illustrate the potential of these materials towithstand the dynamic in vivo environment (eg beating ofthe heart)

34 Network degradation and in vivo tissue response

The in vivo and in vitro mass loss of photopolymerized samplesreached a maximum of sim37 and sim33 respectively at8 weeks (figure 4) The in vivo mass loss was potentiallygreater because of its location in a more dynamic environmentwhere there is more fluid exchange to remove any degradationproducts from the implant region and due to the presenceof enzymes compared to the in vitro environment Alsothe sample preparation was slightly different due to thesterilization of the in vivo samples Based on these massloss data it is suspected that this material would be suitablefor a variety of tissue engineering applications Since redox-intiated samples could not be massed prior to in vivo injectionthe degradation profile could not be monitored However itis anticipated that it would be comparable to its photoinitiatedcounterpart if similar conversions are reached

After two weeks of implantation the host reaction tothe polymer discs comprised granulation tissue with new

(A)

(C )

(B)

(D)

Figure 5 HampE staining for in vivo tissue response to networksformed from macromer 6 at 2 weeks (A) 4 weeks (B) 4 weeks viainjectable redox initiation (C) and 8 weeks (D) (T tissue Ppolymer scale bar = 100 microm)

blood vessels loose connective tissue formation and mildchronic inflammation Macrophages and foreign body giantcells were present at the polymer-tissue interface (figure 5)At the 4 and 8 week time points a thin fibrous capsule ispresent around the implant with minimal associated chronicinflammation (figures 5(B) and (D) respectively) There isno evidence of inflammation or necrosis within the adjacentsubcutaneous fibroadipose tissue skin adnexal structures ordeep skeletal muscle This represents a typical host responseto a biocompatible material The reaction to the injectedpolymer (figure 5(C)) contained slightly more perivascularchronic inflammation in the surrounding host tissue butwithout evidence of necrosis or tissue damage This slightdifference in response to the injected polymer may be due toa mild toxicity associated with the initiators or differences inthe polymer configuration and surface area as compared to thephotoinitiated samples

35 Electrospinning into fibrous scaffolds

Electrospinning has gained much attention in recent yearsas a method for generating fibrous scaffolds [34 35]Fibrous scaffolds are thought to be advantageous since theyclosely mimic the architecture and size-scale of the nativeextracellular matrix [2 34] Many synthetic and naturalpolymers have been successfully electrospun to date [34 37]however the need to electrospin more polymers with a rangeof material properties (eg mechanics and degradation)especially those of elastomers still exists Due to the lowmolecular weight and high polydispersity of Acr-PGS it wasnecessary to modify techniques to electrospin the macromersTan and colleagues recently used photopolymerization and

6

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

(C)

Figure 6 SEM images of electrospun Acr-PGSPEO scaffolds atratios of 3070 (A) 4060 (B) and 5050 (C) Scale bar = 50 microm

electrospinning to create isotropic and anisotropic scaffoldsfrom low molecular weight biodegradable macromers byusing 200 kDa PEO as a carrier polymer [33] Thesame processing protocol was followed to prepare mats ofelectrospun and photocrosslinkable Acr-PGSPEO containinga photoinitiator Mats were electrospun at various ratios ofAcr-PGS to PEO solutions and crosslinked with ultravioletlight prior to visualization under SEM (figure 6) A ratio of3070 PGSPEO (figure 6(A)) produced a mat with the bestmechanical integrity and most distinct fibers which is lostwhen the PEO concentration is too low (figures 6(B) and (C))At this point a thorough characterization of mechanics andcellular interactions has not been performed on these scaffoldsbut our ability to obtain the proper structures motivates furtherexploration of these materials

4 Conclusions

In this study radically polymerized networks with tunablemechanical properties were successfully synthesized andcharacterized Notably an increase in Youngrsquos modulus withincreasing acrylation as well as an increase in the strainat break with increasing molecular weight were observedindicating that these properties can be tuned through the designof the macromer and that networks with elastomeric propertiescan be obtained The reaction behavior was rapid andreached high conversions with both redox and photoinitiatedpolymerizations The networks also degraded more rapidlyin vivo and only mild inflammation was seen even withinjectable formulations Moreover this polymer system couldbe processed into fibrous scaffolds using electrospinning andPEO as a carrier polymer This biodegradable and elastomericpolymer system can be further explored for the engineering

of numerous tissues where elasticity is an importantparameter

Acknowledgments

The authors would like to acknowledge Joshua S Katz forhelpful synthetic discussions and Cindy Chung for assistancewith animal surgeries This work was funded by the AmericanChemical Society Petroleum Research Fund a pilot grant fromthe National Science Foundation Materials Research Scienceand Engineering Center at the University of Pennsylvania andan Ashton Fellowship to JLI

References

[1] Langer R and Vacanti J P 1993 Tissue engineering Science260 920ndash6

[2] Lavik E and Langer R 2004 Tissue engineering current stateand perspectives Appl Microbiol Biotechnol 65 1ndash8

[3] Nerem R M 2006 Tissue engineering the hope the hype andthe future Tissue Eng 12 1143ndash50

[4] Brey D M Ifkovits J L Mozia R I Katz J S and Burdick J A2008 Controlling poly(β-amino ester) network propertiesthrough macromer branching Acta Biomater 4 207ndash17

[5] Chung C Mesa J Randolph M A Yaremchuk M andBurdick J A 2006 Influence of gel properties onneocartilage formation by auricular chondrocytesphotoencapsulated in hyaluronic acid networks J BiomedMater Res A 77 518ndash525

[6] Yang J Webb A R and Ameer G A 2004 Novel citricacid-based biodegradable elastomers for tissue engineeringAdv Mater 16 511

[7] Guan J Stankus J J and Wagner W R 2007 Biodegradableelastomeric scaffolds with basic fibroblast growth factorrelease J Control Release 120 70ndash8

[8] Gunatillake P A and Adhikari R 2003 Biodegradable syntheticpolymers for tissue engineering Eur Cells Mater 5 1ndash16discussion 16

[9] Levental I Georges P C and Janmey P A 2007 Soft biologicalmaterials and their impact on cell function Soft Matter3 299ndash306

[10] Guan J J Sacks M S Beckman E J and Wagner W R 2002Synthesis characterization and cytocompatibility ofefastomeric biodegradable poly(ester-urethane)ureas basedon poly(caprolactone) and putrescine J Biomed MaterRes 61 493ndash503

[11] Ifkovits J L and Burdick J A 2007 Reviewphotopolymerizable and degradable biomaterials for tissueengineering applications Tissue Eng 13 2369ndash85

[12] Anderson D G et al 2006 A combinatorial library ofphotocrosslinkable and degradable materials Adv Mater18 2614

[13] Webb A R Yang J and Ameer G A 2004 Biodegradablepolyester elastomers in tissue engineering Expert OpinBiol Ther 4 801ndash12

[14] Temenoff J S Athanasiou K A LeBaron R G and Mikos A G2002 Effect of poly(ethylene glycol) molecular weight ontensile and swelling properties of oligo(poly(ethyleneglycol) fumarate) hydrogels for cartilage tissue engineeringJ Biomed Mater Res 59 429ndash37

[15] Nuttelman C R Mortisen D J Henry S M and Anseth K S2001 Attachment of fibronectin to poly(vinyl alcohol)hydrogels promotes NIH3T3 cell adhesion proliferationand migration J Biomed Mater Res 57 217ndash23

[16] Wang Y D Ameer G A Sheppard B J and Langer R 2002A tough biodegradable elastomer Nat Biotechnol20 602ndash6

7

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

Figure 2 Consumption of the acrylate peak with time duringphotopolymerization of macromer 1 (05 wt DMPA light intensitysim15 mW cmminus2) (A) Consumption of the acrylate peak with timeduring the redox initiated polymerization of macromer 6 (10 wtBPO and DMPT) (B)

sim8 min The large difference in acrylate absorption betweeninitial and final time points and the near baseline level at thefinal time point indicate a high level of conversion of theacrylate group to crosslinks The redox-initiated crosslinkingwas also monitored by introduction of 10 wt of thebi-component BPO and DMPT initiation system which iscommonly used in bone cements [21ndash23] (figure 2(B)) Themaximum reaction conversion occurred after sim20 min Againthe difference in intensity between the initial and final timepoints indicates high conversion values

The gelation time was defined as the point when a slowlystirring stirbar was stopped after injection of the macromerinto a vial For this system gelation occurred at sim5 minand a maximum temperature of sim30 C (starting from roomtemperature) was observed The maximum conversion andgelation time can be tailored by altering the amount of initiatorincorporated into the macromer system as is the case withsimilar bi-component initiator systems [24 36] dependingon the application For example delivery of this materialto the heart through a catheter might require slower gelationthan direct injection into a defect The minimal increase intemperature is also important if this polymer is to be usedas an injectable formulation to prevent temperature-inducedtissue necrosis

(A)

(B)

Figure 3 Representative tensile stress vs elongation plots fornetworks formed from the Acr-PGS macromers with various MWsand acrylations (A) Youngrsquos modulus (black) and strain at break(white) for networks formed from the various synthesizedmacromers (B) Further information on the macromers can be foundin table 1

33 Network mechanical properties

Polymer slabs for mechanical and degradation analysis wereprepared using photopolymerization Typical tensile stressversus elongation relationships for networks formed fromAcr-PGS macromers are shown in figure 3(A) It is importantto note that many of the samples broke at the clamp andthus could lead to lower than actual values for the strain at break Youngrsquos modulus was determined from theslope of the linear portion of the plot (lt20 strain) andvaried (sim015ndash30 MPa) depending on the Acr-PGS macromer(figure 3(B)) The strain at break also varied (sim5ndash200)depending on the Acr-PGS macromer used for networkformation In general Youngrsquos modulus increased as thedegree of acrylation increased for a given molecular weightAs expected the strain at break increased as Youngrsquosmodulus and acrylation decreased for a given molecularweight Furthermore Youngrsquos modulus and strain atbreak increased with increasing molecular weight for similardegrees of acrylation As seen in figure 3(B) macromers 3and 6 have similar Youngrsquos moduli (sim150 kPa) but their elongation at break varied by almost an order of magnitudeBased on these results it important to note that not all ofthe macromers formed elastomeric networks Therefore

5

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

Figure 4 In vivo (black) and in vitro (white) degradation results at2 4 and 8 weeks for networks formed from macromer 6

macromer 6 was selected for the remaining studies since itsmechanical properties are the most elastomeric

Relationships between macromer structure and networkproperties can easily be drawn from these data For instanceincreased acrylation leads to an increase in the numberof crosslinks formed which is associated with an increasein the modulus of a resulting material and a decrease inthe ability to elongate before failure Additionally moreelastomeric-like features are obtained as the molecular weightof the prepolymer is increased This is a clear demonstrationthat small modifications during synthesis can lead to drasticdifferences in the properties of the resulting material Thusthis same backbone chemistry can be used to develop materialssuitable for a wide range of applications For example a moreelastic and softer material (eg Youngrsquos modulus sim150 kPa)may be more ideal for cardiac tissue engineering whereasa less elastic and a stiffer material (eg Youngrsquos modulussim30 MPa) may be more ideal for bone tissue engineeringAlthough not investigated here dynamic fatigue testing wouldbe necessary to illustrate the potential of these materials towithstand the dynamic in vivo environment (eg beating ofthe heart)

34 Network degradation and in vivo tissue response

The in vivo and in vitro mass loss of photopolymerized samplesreached a maximum of sim37 and sim33 respectively at8 weeks (figure 4) The in vivo mass loss was potentiallygreater because of its location in a more dynamic environmentwhere there is more fluid exchange to remove any degradationproducts from the implant region and due to the presenceof enzymes compared to the in vitro environment Alsothe sample preparation was slightly different due to thesterilization of the in vivo samples Based on these massloss data it is suspected that this material would be suitablefor a variety of tissue engineering applications Since redox-intiated samples could not be massed prior to in vivo injectionthe degradation profile could not be monitored However itis anticipated that it would be comparable to its photoinitiatedcounterpart if similar conversions are reached

After two weeks of implantation the host reaction tothe polymer discs comprised granulation tissue with new

(A)

(C )

(B)

(D)

Figure 5 HampE staining for in vivo tissue response to networksformed from macromer 6 at 2 weeks (A) 4 weeks (B) 4 weeks viainjectable redox initiation (C) and 8 weeks (D) (T tissue Ppolymer scale bar = 100 microm)

blood vessels loose connective tissue formation and mildchronic inflammation Macrophages and foreign body giantcells were present at the polymer-tissue interface (figure 5)At the 4 and 8 week time points a thin fibrous capsule ispresent around the implant with minimal associated chronicinflammation (figures 5(B) and (D) respectively) There isno evidence of inflammation or necrosis within the adjacentsubcutaneous fibroadipose tissue skin adnexal structures ordeep skeletal muscle This represents a typical host responseto a biocompatible material The reaction to the injectedpolymer (figure 5(C)) contained slightly more perivascularchronic inflammation in the surrounding host tissue butwithout evidence of necrosis or tissue damage This slightdifference in response to the injected polymer may be due toa mild toxicity associated with the initiators or differences inthe polymer configuration and surface area as compared to thephotoinitiated samples

35 Electrospinning into fibrous scaffolds

Electrospinning has gained much attention in recent yearsas a method for generating fibrous scaffolds [34 35]Fibrous scaffolds are thought to be advantageous since theyclosely mimic the architecture and size-scale of the nativeextracellular matrix [2 34] Many synthetic and naturalpolymers have been successfully electrospun to date [34 37]however the need to electrospin more polymers with a rangeof material properties (eg mechanics and degradation)especially those of elastomers still exists Due to the lowmolecular weight and high polydispersity of Acr-PGS it wasnecessary to modify techniques to electrospin the macromersTan and colleagues recently used photopolymerization and

6

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

(C)

Figure 6 SEM images of electrospun Acr-PGSPEO scaffolds atratios of 3070 (A) 4060 (B) and 5050 (C) Scale bar = 50 microm

electrospinning to create isotropic and anisotropic scaffoldsfrom low molecular weight biodegradable macromers byusing 200 kDa PEO as a carrier polymer [33] Thesame processing protocol was followed to prepare mats ofelectrospun and photocrosslinkable Acr-PGSPEO containinga photoinitiator Mats were electrospun at various ratios ofAcr-PGS to PEO solutions and crosslinked with ultravioletlight prior to visualization under SEM (figure 6) A ratio of3070 PGSPEO (figure 6(A)) produced a mat with the bestmechanical integrity and most distinct fibers which is lostwhen the PEO concentration is too low (figures 6(B) and (C))At this point a thorough characterization of mechanics andcellular interactions has not been performed on these scaffoldsbut our ability to obtain the proper structures motivates furtherexploration of these materials

4 Conclusions

In this study radically polymerized networks with tunablemechanical properties were successfully synthesized andcharacterized Notably an increase in Youngrsquos modulus withincreasing acrylation as well as an increase in the strainat break with increasing molecular weight were observedindicating that these properties can be tuned through the designof the macromer and that networks with elastomeric propertiescan be obtained The reaction behavior was rapid andreached high conversions with both redox and photoinitiatedpolymerizations The networks also degraded more rapidlyin vivo and only mild inflammation was seen even withinjectable formulations Moreover this polymer system couldbe processed into fibrous scaffolds using electrospinning andPEO as a carrier polymer This biodegradable and elastomericpolymer system can be further explored for the engineering

of numerous tissues where elasticity is an importantparameter

Acknowledgments

The authors would like to acknowledge Joshua S Katz forhelpful synthetic discussions and Cindy Chung for assistancewith animal surgeries This work was funded by the AmericanChemical Society Petroleum Research Fund a pilot grant fromthe National Science Foundation Materials Research Scienceand Engineering Center at the University of Pennsylvania andan Ashton Fellowship to JLI

References

[1] Langer R and Vacanti J P 1993 Tissue engineering Science260 920ndash6

[2] Lavik E and Langer R 2004 Tissue engineering current stateand perspectives Appl Microbiol Biotechnol 65 1ndash8

[3] Nerem R M 2006 Tissue engineering the hope the hype andthe future Tissue Eng 12 1143ndash50

[4] Brey D M Ifkovits J L Mozia R I Katz J S and Burdick J A2008 Controlling poly(β-amino ester) network propertiesthrough macromer branching Acta Biomater 4 207ndash17

[5] Chung C Mesa J Randolph M A Yaremchuk M andBurdick J A 2006 Influence of gel properties onneocartilage formation by auricular chondrocytesphotoencapsulated in hyaluronic acid networks J BiomedMater Res A 77 518ndash525

[6] Yang J Webb A R and Ameer G A 2004 Novel citricacid-based biodegradable elastomers for tissue engineeringAdv Mater 16 511

[7] Guan J Stankus J J and Wagner W R 2007 Biodegradableelastomeric scaffolds with basic fibroblast growth factorrelease J Control Release 120 70ndash8

[8] Gunatillake P A and Adhikari R 2003 Biodegradable syntheticpolymers for tissue engineering Eur Cells Mater 5 1ndash16discussion 16

[9] Levental I Georges P C and Janmey P A 2007 Soft biologicalmaterials and their impact on cell function Soft Matter3 299ndash306

[10] Guan J J Sacks M S Beckman E J and Wagner W R 2002Synthesis characterization and cytocompatibility ofefastomeric biodegradable poly(ester-urethane)ureas basedon poly(caprolactone) and putrescine J Biomed MaterRes 61 493ndash503

[11] Ifkovits J L and Burdick J A 2007 Reviewphotopolymerizable and degradable biomaterials for tissueengineering applications Tissue Eng 13 2369ndash85

[12] Anderson D G et al 2006 A combinatorial library ofphotocrosslinkable and degradable materials Adv Mater18 2614

[13] Webb A R Yang J and Ameer G A 2004 Biodegradablepolyester elastomers in tissue engineering Expert OpinBiol Ther 4 801ndash12

[14] Temenoff J S Athanasiou K A LeBaron R G and Mikos A G2002 Effect of poly(ethylene glycol) molecular weight ontensile and swelling properties of oligo(poly(ethyleneglycol) fumarate) hydrogels for cartilage tissue engineeringJ Biomed Mater Res 59 429ndash37

[15] Nuttelman C R Mortisen D J Henry S M and Anseth K S2001 Attachment of fibronectin to poly(vinyl alcohol)hydrogels promotes NIH3T3 cell adhesion proliferationand migration J Biomed Mater Res 57 217ndash23

[16] Wang Y D Ameer G A Sheppard B J and Langer R 2002A tough biodegradable elastomer Nat Biotechnol20 602ndash6

7

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

Figure 4 In vivo (black) and in vitro (white) degradation results at2 4 and 8 weeks for networks formed from macromer 6

macromer 6 was selected for the remaining studies since itsmechanical properties are the most elastomeric

Relationships between macromer structure and networkproperties can easily be drawn from these data For instanceincreased acrylation leads to an increase in the numberof crosslinks formed which is associated with an increasein the modulus of a resulting material and a decrease inthe ability to elongate before failure Additionally moreelastomeric-like features are obtained as the molecular weightof the prepolymer is increased This is a clear demonstrationthat small modifications during synthesis can lead to drasticdifferences in the properties of the resulting material Thusthis same backbone chemistry can be used to develop materialssuitable for a wide range of applications For example a moreelastic and softer material (eg Youngrsquos modulus sim150 kPa)may be more ideal for cardiac tissue engineering whereasa less elastic and a stiffer material (eg Youngrsquos modulussim30 MPa) may be more ideal for bone tissue engineeringAlthough not investigated here dynamic fatigue testing wouldbe necessary to illustrate the potential of these materials towithstand the dynamic in vivo environment (eg beating ofthe heart)

34 Network degradation and in vivo tissue response

The in vivo and in vitro mass loss of photopolymerized samplesreached a maximum of sim37 and sim33 respectively at8 weeks (figure 4) The in vivo mass loss was potentiallygreater because of its location in a more dynamic environmentwhere there is more fluid exchange to remove any degradationproducts from the implant region and due to the presenceof enzymes compared to the in vitro environment Alsothe sample preparation was slightly different due to thesterilization of the in vivo samples Based on these massloss data it is suspected that this material would be suitablefor a variety of tissue engineering applications Since redox-intiated samples could not be massed prior to in vivo injectionthe degradation profile could not be monitored However itis anticipated that it would be comparable to its photoinitiatedcounterpart if similar conversions are reached

After two weeks of implantation the host reaction tothe polymer discs comprised granulation tissue with new

(A)

(C )

(B)

(D)

Figure 5 HampE staining for in vivo tissue response to networksformed from macromer 6 at 2 weeks (A) 4 weeks (B) 4 weeks viainjectable redox initiation (C) and 8 weeks (D) (T tissue Ppolymer scale bar = 100 microm)

blood vessels loose connective tissue formation and mildchronic inflammation Macrophages and foreign body giantcells were present at the polymer-tissue interface (figure 5)At the 4 and 8 week time points a thin fibrous capsule ispresent around the implant with minimal associated chronicinflammation (figures 5(B) and (D) respectively) There isno evidence of inflammation or necrosis within the adjacentsubcutaneous fibroadipose tissue skin adnexal structures ordeep skeletal muscle This represents a typical host responseto a biocompatible material The reaction to the injectedpolymer (figure 5(C)) contained slightly more perivascularchronic inflammation in the surrounding host tissue butwithout evidence of necrosis or tissue damage This slightdifference in response to the injected polymer may be due toa mild toxicity associated with the initiators or differences inthe polymer configuration and surface area as compared to thephotoinitiated samples

35 Electrospinning into fibrous scaffolds

Electrospinning has gained much attention in recent yearsas a method for generating fibrous scaffolds [34 35]Fibrous scaffolds are thought to be advantageous since theyclosely mimic the architecture and size-scale of the nativeextracellular matrix [2 34] Many synthetic and naturalpolymers have been successfully electrospun to date [34 37]however the need to electrospin more polymers with a rangeof material properties (eg mechanics and degradation)especially those of elastomers still exists Due to the lowmolecular weight and high polydispersity of Acr-PGS it wasnecessary to modify techniques to electrospin the macromersTan and colleagues recently used photopolymerization and

6

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

(C)

Figure 6 SEM images of electrospun Acr-PGSPEO scaffolds atratios of 3070 (A) 4060 (B) and 5050 (C) Scale bar = 50 microm

electrospinning to create isotropic and anisotropic scaffoldsfrom low molecular weight biodegradable macromers byusing 200 kDa PEO as a carrier polymer [33] Thesame processing protocol was followed to prepare mats ofelectrospun and photocrosslinkable Acr-PGSPEO containinga photoinitiator Mats were electrospun at various ratios ofAcr-PGS to PEO solutions and crosslinked with ultravioletlight prior to visualization under SEM (figure 6) A ratio of3070 PGSPEO (figure 6(A)) produced a mat with the bestmechanical integrity and most distinct fibers which is lostwhen the PEO concentration is too low (figures 6(B) and (C))At this point a thorough characterization of mechanics andcellular interactions has not been performed on these scaffoldsbut our ability to obtain the proper structures motivates furtherexploration of these materials

4 Conclusions

In this study radically polymerized networks with tunablemechanical properties were successfully synthesized andcharacterized Notably an increase in Youngrsquos modulus withincreasing acrylation as well as an increase in the strainat break with increasing molecular weight were observedindicating that these properties can be tuned through the designof the macromer and that networks with elastomeric propertiescan be obtained The reaction behavior was rapid andreached high conversions with both redox and photoinitiatedpolymerizations The networks also degraded more rapidlyin vivo and only mild inflammation was seen even withinjectable formulations Moreover this polymer system couldbe processed into fibrous scaffolds using electrospinning andPEO as a carrier polymer This biodegradable and elastomericpolymer system can be further explored for the engineering

of numerous tissues where elasticity is an importantparameter

Acknowledgments

The authors would like to acknowledge Joshua S Katz forhelpful synthetic discussions and Cindy Chung for assistancewith animal surgeries This work was funded by the AmericanChemical Society Petroleum Research Fund a pilot grant fromthe National Science Foundation Materials Research Scienceand Engineering Center at the University of Pennsylvania andan Ashton Fellowship to JLI

References

[1] Langer R and Vacanti J P 1993 Tissue engineering Science260 920ndash6

[2] Lavik E and Langer R 2004 Tissue engineering current stateand perspectives Appl Microbiol Biotechnol 65 1ndash8

[3] Nerem R M 2006 Tissue engineering the hope the hype andthe future Tissue Eng 12 1143ndash50

[4] Brey D M Ifkovits J L Mozia R I Katz J S and Burdick J A2008 Controlling poly(β-amino ester) network propertiesthrough macromer branching Acta Biomater 4 207ndash17

[5] Chung C Mesa J Randolph M A Yaremchuk M andBurdick J A 2006 Influence of gel properties onneocartilage formation by auricular chondrocytesphotoencapsulated in hyaluronic acid networks J BiomedMater Res A 77 518ndash525

[6] Yang J Webb A R and Ameer G A 2004 Novel citricacid-based biodegradable elastomers for tissue engineeringAdv Mater 16 511

[7] Guan J Stankus J J and Wagner W R 2007 Biodegradableelastomeric scaffolds with basic fibroblast growth factorrelease J Control Release 120 70ndash8

[8] Gunatillake P A and Adhikari R 2003 Biodegradable syntheticpolymers for tissue engineering Eur Cells Mater 5 1ndash16discussion 16

[9] Levental I Georges P C and Janmey P A 2007 Soft biologicalmaterials and their impact on cell function Soft Matter3 299ndash306

[10] Guan J J Sacks M S Beckman E J and Wagner W R 2002Synthesis characterization and cytocompatibility ofefastomeric biodegradable poly(ester-urethane)ureas basedon poly(caprolactone) and putrescine J Biomed MaterRes 61 493ndash503

[11] Ifkovits J L and Burdick J A 2007 Reviewphotopolymerizable and degradable biomaterials for tissueengineering applications Tissue Eng 13 2369ndash85

[12] Anderson D G et al 2006 A combinatorial library ofphotocrosslinkable and degradable materials Adv Mater18 2614

[13] Webb A R Yang J and Ameer G A 2004 Biodegradablepolyester elastomers in tissue engineering Expert OpinBiol Ther 4 801ndash12

[14] Temenoff J S Athanasiou K A LeBaron R G and Mikos A G2002 Effect of poly(ethylene glycol) molecular weight ontensile and swelling properties of oligo(poly(ethyleneglycol) fumarate) hydrogels for cartilage tissue engineeringJ Biomed Mater Res 59 429ndash37

[15] Nuttelman C R Mortisen D J Henry S M and Anseth K S2001 Attachment of fibronectin to poly(vinyl alcohol)hydrogels promotes NIH3T3 cell adhesion proliferationand migration J Biomed Mater Res 57 217ndash23

[16] Wang Y D Ameer G A Sheppard B J and Langer R 2002A tough biodegradable elastomer Nat Biotechnol20 602ndash6

7

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

(A)

(B)

(C)

Figure 6 SEM images of electrospun Acr-PGSPEO scaffolds atratios of 3070 (A) 4060 (B) and 5050 (C) Scale bar = 50 microm

electrospinning to create isotropic and anisotropic scaffoldsfrom low molecular weight biodegradable macromers byusing 200 kDa PEO as a carrier polymer [33] Thesame processing protocol was followed to prepare mats ofelectrospun and photocrosslinkable Acr-PGSPEO containinga photoinitiator Mats were electrospun at various ratios ofAcr-PGS to PEO solutions and crosslinked with ultravioletlight prior to visualization under SEM (figure 6) A ratio of3070 PGSPEO (figure 6(A)) produced a mat with the bestmechanical integrity and most distinct fibers which is lostwhen the PEO concentration is too low (figures 6(B) and (C))At this point a thorough characterization of mechanics andcellular interactions has not been performed on these scaffoldsbut our ability to obtain the proper structures motivates furtherexploration of these materials

4 Conclusions

In this study radically polymerized networks with tunablemechanical properties were successfully synthesized andcharacterized Notably an increase in Youngrsquos modulus withincreasing acrylation as well as an increase in the strainat break with increasing molecular weight were observedindicating that these properties can be tuned through the designof the macromer and that networks with elastomeric propertiescan be obtained The reaction behavior was rapid andreached high conversions with both redox and photoinitiatedpolymerizations The networks also degraded more rapidlyin vivo and only mild inflammation was seen even withinjectable formulations Moreover this polymer system couldbe processed into fibrous scaffolds using electrospinning andPEO as a carrier polymer This biodegradable and elastomericpolymer system can be further explored for the engineering

of numerous tissues where elasticity is an importantparameter

Acknowledgments

The authors would like to acknowledge Joshua S Katz forhelpful synthetic discussions and Cindy Chung for assistancewith animal surgeries This work was funded by the AmericanChemical Society Petroleum Research Fund a pilot grant fromthe National Science Foundation Materials Research Scienceand Engineering Center at the University of Pennsylvania andan Ashton Fellowship to JLI

References

[1] Langer R and Vacanti J P 1993 Tissue engineering Science260 920ndash6

[2] Lavik E and Langer R 2004 Tissue engineering current stateand perspectives Appl Microbiol Biotechnol 65 1ndash8

[3] Nerem R M 2006 Tissue engineering the hope the hype andthe future Tissue Eng 12 1143ndash50

[4] Brey D M Ifkovits J L Mozia R I Katz J S and Burdick J A2008 Controlling poly(β-amino ester) network propertiesthrough macromer branching Acta Biomater 4 207ndash17

[5] Chung C Mesa J Randolph M A Yaremchuk M andBurdick J A 2006 Influence of gel properties onneocartilage formation by auricular chondrocytesphotoencapsulated in hyaluronic acid networks J BiomedMater Res A 77 518ndash525

[6] Yang J Webb A R and Ameer G A 2004 Novel citricacid-based biodegradable elastomers for tissue engineeringAdv Mater 16 511

[7] Guan J Stankus J J and Wagner W R 2007 Biodegradableelastomeric scaffolds with basic fibroblast growth factorrelease J Control Release 120 70ndash8

[8] Gunatillake P A and Adhikari R 2003 Biodegradable syntheticpolymers for tissue engineering Eur Cells Mater 5 1ndash16discussion 16

[9] Levental I Georges P C and Janmey P A 2007 Soft biologicalmaterials and their impact on cell function Soft Matter3 299ndash306

[10] Guan J J Sacks M S Beckman E J and Wagner W R 2002Synthesis characterization and cytocompatibility ofefastomeric biodegradable poly(ester-urethane)ureas basedon poly(caprolactone) and putrescine J Biomed MaterRes 61 493ndash503

[11] Ifkovits J L and Burdick J A 2007 Reviewphotopolymerizable and degradable biomaterials for tissueengineering applications Tissue Eng 13 2369ndash85

[12] Anderson D G et al 2006 A combinatorial library ofphotocrosslinkable and degradable materials Adv Mater18 2614

[13] Webb A R Yang J and Ameer G A 2004 Biodegradablepolyester elastomers in tissue engineering Expert OpinBiol Ther 4 801ndash12

[14] Temenoff J S Athanasiou K A LeBaron R G and Mikos A G2002 Effect of poly(ethylene glycol) molecular weight ontensile and swelling properties of oligo(poly(ethyleneglycol) fumarate) hydrogels for cartilage tissue engineeringJ Biomed Mater Res 59 429ndash37

[15] Nuttelman C R Mortisen D J Henry S M and Anseth K S2001 Attachment of fibronectin to poly(vinyl alcohol)hydrogels promotes NIH3T3 cell adhesion proliferationand migration J Biomed Mater Res 57 217ndash23

[16] Wang Y D Ameer G A Sheppard B J and Langer R 2002A tough biodegradable elastomer Nat Biotechnol20 602ndash6

7

Biomed Mater 3 (2008) 034104 J L Ifkovits et al

[17] Gerecht S et al 2007 A porous photocurable elastomer for cellencapsulation and culture Biomaterials 28 4826ndash35

[18] Nijst C L et al 2007 Synthesis and characterization ofphotocurable elastomers from poly(glycerol-co-sebacate)Biomacromolecules 8 3067ndash73

[19] Gao J Crapo P M and Wang Y D 2006 Macroporouselastomeric scaffolds with extensive micropores for softtissue engineering Tissue Eng 12 917ndash25

[20] Odian G 2004 Principles of Polymerization 4th edn (HobokenNJ Wiley)

[21] Basgorenay B Ulubayram K Serbetci K Onurhan E andHasirci N 2006 Preparation modification andcharacterization of acrylic cements J Appl Polym Sci99 3631ndash7

[22] Nussbaum D A Gailloud P and Murphy K 2004 The chemistryof acrylic bone cements and implications for clinical use inimage-guided therapy J Vasc Interv Radiol 15 121ndash6

[23] Punyani S Deb S and Singh H 2007 Contact killingantimicrobial acrylic bone cements preparation andcharacterization J Biomater Sci Polym Ed 18 131ndash45

[24] Duan S F Zhu W Yu L and Ding J D 2005 Negativecooperative effect of cytotoxicity of a di-componentinitiating system for a novel injectable tissue engineeringhydrogel Chin Sci Bull 50 1093ndash6

[25] Bonzani I C Adhikari R Houshyar S Mayadunne RGunatillake P and Stevens M M 2007 Synthesis oftwo-component injectable polyurethanes for bone tissueengineering Biomaterials 28 423ndash33

[26] Akala E O Elekwachi O and Obidi A 2003 Studies on butylacrylate-based hydrogels fabricated byorganic-redox-initiated polymerization process for thedelivery of thermolabile bioactive agents Pharm Ind65 1075ndash81

[27] Christman K L Fok H H Sievers R E Fang Q H and Lee R J2004 Fibrin glue alone and skeletal myoblasts in a fibrinscaffold preserve cardiac function after myocardialinfarction Tissue Eng 10 403ndash9

[28] Kofidis T Lebl D R Martinez E C Hoyt G Tanaka M andRobbins R 2004 Novel injectable bioartificial tissuefacilitates targeted less invasive large-scale tissuerestoration following myocardial injury Circulation110 508

[29] Burdick J A Peterson A J and Anseth K S 2001 Conversionand temperature profiles during the photoinitiatedpolymerization of thick orthopaedic biomaterialsBiomaterials 22 1779ndash86

[30] Albrecht D R Tsang V L Sah R L and Bhatia S N 2005Photo- and electropatterning of hydrogel-encapsulatedliving cell arrays Lab Chip 5 111ndash8

[31] Gopalan S M et al 2003 Anisotropic stretch-inducedhypertrophy in neonatal ventricular myocytesmicropatterned on deformable elastomers BiotechnolBioeng 81 578ndash87

[32] Tsang V L et al 2007 Fabrication of 3D hepatic tissues byadditive photopatterning of cellular hydrogels Faseb J21 790ndash801

[33] Tan A R Ifkovits J L Baker B M Brey D M Mauck R L andBurdick J A 2008 Electrospinning of photocrosslinked anddegradable fibrous scaffolds J Biomed Mater Resdoi101002jbma31853

[34] Liao S Li B J Ma Z W Wei H Chan C and Ramakrishna S2006 Biomimetic electrospun nanofibers for tissueregeneration Biomed Mater 1 R45ndash53

[35] Fridrikh S V Yu J H Brenner M P and Rutledge G C 2003Controlling the fiber diameter during electrospinning PhysRev Lett 90 144502

[36] Temenoff J S Shin H Conway D E Engel P S andMikos A G 2003 In vitro cytotoxicity of redox radicalinitiators for cross-linking of oligo(poly(ethylene glycol)fumarate) macromers Biomacromolecules4 1605ndash13

[37] Murugan R and Ramakrishna S 2006 Nano-featured scaffoldsfor tissue engineering A review of spinning methodologiesTissue Eng 12 435ndash47

8