Influence of Cross-Linking on the Physical Properties and Cytotoxicity of Polyhydroxyalkanoate (PHA)...

Influence of Cross-Linking on the Physical Properties andCytotoxicity of Polyhydroxyalkanoate (PHA) Scaffolds for TissueEngineeringAlex C. Levine,† Angelina Sparano,† Frederick F. Twigg,† Keiji Numata,‡ and Christopher T. Nomura*,†,§

†Department of Chemistry and §Center for Applied Microbiology, State University of New York−College of Environmental Scienceand Forestry, 1 Forestry Drive, Syracuse, New York 13210, United States‡Enzyme Research Team, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

ABSTRACT: In this study, an unsaturated copolyester, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxy-10-undecenoate](PHBU), was produced by an engineered strain of Escherichia coli, cross-linked via thiol-ene click chemistry, and analyzed forimproved physical properties and biocompatibility with human mesenchymal stem cells. By cross-linking the PHBU polymer, anincrease in tensile strength of greater than 200% to 26.2 MPa was observed, resulting in a material with physical properties closerto those relevant for soft tissue replacement. Results showed that this chemically cross-linked polyester did not exhibit significantcytotoxicity toward human cells after chemical modification. The chemically modifiable copolyester described here couldpotentially be used as a replacement for an assortment of tissues currently without viable material alternatives in the field oftissue-engineering.

KEYWORDS: polyhydroxyalkanoates, biomaterials, Escherichia coli, cross-linking, thiol-ene click chemistry,human mesenchymal stem cells, tissue engineering, scaffold

■ INTRODUCTIONBiomedical scaffolds are materials that are used to replace orregenerate living tissue1 by providing support for cellattachment and growth. Because of the great variety of celltypes and complex nature of cell attachment,2 an ideal scaffoldmaterial should be tailorable to encourage the production of ormimic the extracellular matrix of target tissues3 in order tofacilitate compatibility and growth and, in the case of stem cells,differentiation.4 The main considerations when selecting theappropriate material for a tissue-engineering scaffold arearchitecture, tissue/cyto-compatibility, bioactivity, and mechan-ical properties.1,5 The architecture of the scaffold should behighly porous to allow transport of nutrients and vasculariza-tion, although not so porous as to compromise the strength ofthe material.6,7,6 The scaffold should also biodegrade in vivowhile simultaneously being replaced by cells of the targettissue.8 To support cell attachment, growth, and proliferation,the scaffold material must be compatible with cell types andcomponents both in vitro and in vivo.9 In vitro compatibility isrelevant when seeding the scaffolds with cells prior to

implantation and in vivo compatibility is crucial to preventimmune response and rejection of the implant. The bioactivityof the scaffolds must also be considered, as cell attachment andmorphology can be effected by the scaffold topology.2 Inaddition to surface chemistry, the smoothness or roughness ofthe scaffold surface can greatly affect cellular attachment. If thescaffold is serving as a delivery method for biological signals tostimulate growth,10 then the release of the compound must bestringently controlled for therapeutic efficacy and nontoxicity.Finally, the mechanical properties of the scaffold must matchthat of the target tissue.3 Scaffolds with mismatchingmechanical stiffness or elasticity will have decreased perform-ance in situ and the implant itself may be prematurely degradedor irreparably damaged.11,12

Polymers used for tissue-engineered scaffolds vary greatly,and include both synthetic and natural polymers such as

Received: January 31, 2015Accepted: May 27, 2015

Article

pubs.acs.org/journal/abseba

© XXXX American Chemical Society A DOI: 10.1021/acsbiomaterials.5b00052ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

proteins, polysaccharides, and polyesters.13,14 Biological poly-mers are of great interest as tissue engineering materials as theyhave greater biocompatibility and biodegradability compared tosynthetic polymers, though the mechanical properties of thesebiopolymers are often inferior to synthetic materials. Collagenis a well-studied biopolymer in tissue-engineering due to itsbioactivity and inherent biocompatibility.15,16 However,collagen alone cannot always meet the mechanical require-ments for tissue replacement, and it is common for collagen tobe blended with other polymers to create compositematerials.17 In these composite materials, the role of collagenis to increase bioactivity and cellular attachment, whereas theother purpose of the other polymer is to provide suitablestrength to the material to prevent damage and improveapplication. Collagen composites with polylactic acid, a type ofbiopolyester, have been used for vascular grafts where thecollagen provides a surface that cells can readily attach to, andthe biopolyester serves to maintain the shape of the tissue.18

Polysaccharides such as cellulose are also of interest, and it hasrecently been shown that the native cell-wall structure in applescan be used as a tissue scaffold.19 In the realm of polyesters,polylactic acid (PLA),20 polycaprolactone (PCL),13,21 andpolyhydroxyalkanoates (PHA)22−24 have been-well studied.PHAs are of particular interest as they have a greater range ofphysical properties as compared to other biopolyesters, withapplications such as heart valves already showing promisingresults in animal models.25

Previous studies have demonstrated the effect of repeatingunit composition on the material properties of PHA polymersand have shown that PHA polymers comprised of short-chain-length (SCL) repeating units of 3−5 carbons behave asthermoplastics and PHA polymers comprised of medium-chain-length (MCL) repeating units of 6−14 carbons behave aselastomers. Copolymers of SCL and MCL repeating units havephysical properties ranging from SCL to MCL homopolymers.In addition, by controlling the ratio of SCL to MCL repeatingunits, the material properties can cover a wide range of physicalproperties.26−29 Recently our lab engineered a strain Escherichiacoli LSBJ (E. coli LSBJ) to produce PHA homopolymers andcopolymers with defined repeating compositions. Strictlydefining the repeating unit composition of PHA polymersand copolymers allows for stringent control over PHA physicalproperties such as mechanical strength, thermal properties, andhydrophobicity.28,29 These PHA materials may be suited fortissue-engineering scaffolds based on the required stiffness forthe application and biodegradability. Additionally, for the firsttime, PHA polymers produced from this strain of bacteria canbe programmed to contain specific quantities of olefinfunctional groups, which can be subsequently modified viasynthetic chemistry to provide further diversification of materialproperties. Although other works have shown the potential tochemically modify PHAs,30,31 these previous studies were notfocused on tissue-engineering applications and used PHApolymers where repeating unit compositions were highlyvariable because of the native metabolism of the bacterialstrain used to produce said polymers.The goal of this work was to produce a strong and flexible

PHA scaffold of PHA with inherent biocompatibility.30,32,33 Inthis work, we have produced and characterized a new PHAcopolymer for tissue engineering scaffolds, poly[(R)-3-hydrox-ybutyrate-co-(R)-3-hydroxy-10-undecenoate] (PHBU). PHBUis a short-chain-length/medium-chain-length (SCL/MCL)PHA copolymer that contains a terminal alkene functional

group in the side chain of its MCL repeating unit. This degreeof unsaturation was used as a site to strategically improve thephysical properties of the PHBU scaffolds using thiol-ene clickchemistry. The benefits to the physical properties of polymersvia cross-linking have been well established,34,35 with observedimprovements to tensile strength, stiffness, and cell attachment.PHA cross-linked by thiol-ene click chemistry has tailorablephysical characteristics dependent on the density of cross-linking, and can be controlled by varying the concentration ofthe cross-linker. The work presented here demonstrates thatthiol-ene click chemistry can be employed to chemically cross-link PHA, resulting in a PHA scaffold with significantlyenhanced tensile strength and insignificant effects oncytotoxicity. It is anticipated that this cross-linked PHA scaffoldwill find use in tissue-engineered scaffolds that require stiffermaterials, such as cartilage and ligament replacements.34−37

■ EXPERIMENTAL SECTIONPoly[(R)-3-Hydroxybutyrate-co-(R)-3hydroxy-10-undece-

noate] (PHBU) Production Using E. coli LSBJ. The SCL/MCLcopolymer PHBU was produced as described for poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyoctanoate] by Tappel et. al.29 Thestrain used for polymer production was E. coli LSBJ, a derivative of E.coli LS5218 with deletion of genes fadB and fadJ, and harboring aplasmid containing the genes for an (R)-specific enoyl-CoA hydratase(phaJ4) and PHA synthase [phaC1(STQK)].29 Starter culturesconsisted of a 250 mL Erlenmeyer flask containing 50 mL of Lennoxbroth (LB) and 50 mg/L kanamycin, which was inoculated with E. coliLSBJ and incubated for 16−18 h at 30 °C in an orbital shaker set at250 rpm. For polymer production, 1 mL of this culture was used toinoculate 500 mL of baffled flasks containing 100 mL of LB medium,50 mg/L kanamycin, 4 g/L Brij-35 (surfactant), and 2 g/L fatty acids.Cultures were grown at 30 °C for 48 h in an orbital shaker set at 250rpm. Following, cells were harvested with centrifugation for 15 min at3716 × g and 22 °C, and the supernatant was discarded. Cell pelletswere washed with 70% ethanol to remove residual fatty acids, collectedagain by centrifugation under the same conditions, and washed withnanopure water. After being washed with water, the cells werecollected by centrifugation once more and resuspended in 15 mL ofwater before freeze-drying. After drying by lyophilization, the cellswere suspended in methanol (22 mL/g dried cell mass) and gentlystirred for 5 min at 22 °C. Afterward, cells were collected bycentrifugation and washed with nanopure water. Cells were again driedby lyophilization, and PHAs were purified via Soxhlet extractionfollowed by methanol precipitation. Soxhlet extractions wereperformed in 120 mL of chloroform for 5 h, and then the solutionwas transferred to a glass Petri dish where the solvent was evaporatedunder ambient conditions for >6 h. The resulting film was dissolved ina minimal amount of chloroform, and precipitated into a 10-fold largervolume of methanol. The final white powder was stored at 22 °C inthe dark until scaffold fabrication.

Molecular Weight and Repeating Unit Content Determi-nation. Molecular weight was determined using a Shimadzu GPCsystem. Samples were prepared for GPC by transferring 1−2 mg ofpolymer to a 5 mL glass vial and dissolving the material in chloroformto a final concentration of 0.7 mg/mL by heating at 50 °C. Oncedissolved, 1 mL of solution was filtered through a 0.45 μm PTFEsyringe filter into a 2 mL GPC vial. Sample volumes of 50 μL wereinjected into a LC-20AD liquid chromatograph equipped with a SIL-20A autosampler, CTO-20A column oven, and an RID-10A refractiveindex detector (Shimadzu). Chromatography was performed using a 8× 50 mm styrenedivinylbenzene (SDV) guard column (5 μm particles;Polymer Standards Service) and a 8 × 300 mm SDV analytical column(5 μm particles; mixed bed porosity; max molecular weight 1E6 Da;Polymer Standards Service product sda083005lim). The mobile phaseconsisted of chloroform at a flow rate of 1 mL/min and thetemperature was maintained at 40 °C. Analysis was performed usingShimadzu LCsolution software. The repeating unit content of PHBU

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was determined by 1HNMR spectroscopy using a Bruker AVANCE600 spectrometer. Samples consisting of 15 mg of polymer weredissolved in 1 mL of deuterated chloroform. Spectra were processedusing TOSPIN v1.3 from Bruker BioSpin. The ratio of the alkeneproton signal at 5.8 ppm to the polymer backbone stereocenter at 5.3ppm was taken as the MCL portion of the copolymer. A series ofPHBU copolymers with varying MCL content were generated toproduce a standard curve for precise PHBU synthesis. From this data,a PHBU copolymer with 8% MCL was produced for scaffoldfabrication.PHA Scaffold Fabrication. PHA scaffolds were fabricated using a

combined method of salt-leaching20,21,40 and thermally induced phaseseparation.1,10,40 To a 3 mL glass vial were added 0.10 g of PHBU (8.4× 10−5 moles of alkene) and 1 mL of 1,4-dioxane, and the polymer wasdissolved by stirring and heating at 60 °C for 30 min. To this solution,1.0 g of sodium chloride was added, and the resulting heterogeneousmixture was transferred to a custom-made mold consisting of twomicroscope slides separated by a 1 mm thick Teflon spacer. Thedimensions of the mold’s chamber were 60, 25, and 1 mm (L × W ×T). The samples were frozen at −80 °C for 1 h, and then the solventremoved by lyophilization for 24 h. The resulting PHBU/saltcomposite was placed in 300 mL Milli-Q water with stirring at 25°C for 4 h, with complete water changes after each hour. Thecomposite was soaked for an additional 48 h, with water changes every24 h. The resulting PHBU scaffold was dried under vacuum for 48 h.PHBU Scaffold Cross-Linking. PHBU contains a terminal alkene

functional group as part of the polymer side-chain. This site can bechemically modified via thiol-ene click chemistry.41 Thiol-ene clickchemistry is a radical mediated coupling reaction between alkene andthiol functional groups, resulting in a covalent thioether linkage in afast, efficient, and water/oxygen insensitive reaction. The PHBUcopolymer was cross-linked using pentaerythritol tetrakis (3-mercaptopropionate) (PETMP) and radical initiator 2,2-dimethox-yphenylacetophenone (DMPA), both purchased from Sigma-Aldrich.For fully cross-linked samples, 8.0 μL of PETMP (2.1 × 10−5 moles)and 1 mg of DMPA were added to the PHBU solution prior totransfer into the mold. The amount of PETMP was chosen to be 25%the concentration of available alkene functional groups in PHBU dueto PETMP’s four thiol functional groups. This rationale was based onthe criteria of maximizing cross-link density and reducing the amountof unreacted PETMP in the PHBU scaffolds. Samples were also madeusing half PETMP concentration at 4.0 μL. After transferring thepolymer solution, the mold was placed under a UVP UVGL-58 hand-held lamp and irradiated with 365 nm UV light for 1 h. Followingirradiation, the samples were frozen and treated identically to non-cross-linked samples with treatment by lyophilization, salt leaching,and drying.Assessment of Cross-Linking by Fourier Transform Infrared

Spectroscopy (FT-IR). FT-IR was utilized to determine the degree ofcross-linking in the PHBU polymer by measuring the change instretching vibration at 1641 cm−1, which corresponded to the alkenefunctional group. The reduction of this signal was taken as evidencefor formation of new thioether linkages via the thiol-ene click reaction.Data were collected using a Bruker Tensor 27 spectrometer equippedwith an attenuated total reflection (ATR) stage. Each sample received32 scans and data was extracted in transmittance mode with OPUS 6.5software. Data was replotted using Microsoft Excel 2007.Gel-Soluble Fraction. To determine the extent of the cross-linked

network within the scaffolds, we measured the gel fraction35 of eachsample after incubation in chloroform. PHBU samples of 5−10 mgwere weighed, and each placed into a 3 mL glass vial containing 2 mLof chloroform. After incubation at 25 °C for 48 h, the solvent wasremoved, and the swollen PHA polymer was rinsed with a fresh 1 mLportion of chloroform. At this time, the cross-linked portion of thePHA scaffold was dried under vacuum for 48 h. The gel fraction wasdetermined by

= ·G W W/ 100f f 0

Where Gf is the weight percentage of cross-linked PHBU, Wf is theweight of the sample after chloroform extraction, and W0 is the initialweight of the sample.

Cell Culture and Viability. To assess compatibility with livingcells, human mesenchymal stem cells (hMSC),4 a type of multipotentstromal cell able to differentiate into a variety of cell types, were usedto seed the PHBU scaffolds. To determine whether cross-linking hadan effect on cytotoxicity, cultures of hMSC seeded onto the PHBUscaffolds were examined. The scaffolds were cut into 5 × 5 × 1 mmsquares, and attached to the bottom of the wells in a polystyrene 96-well plate using a small amount of chloroform, and dried undervacuum for 48 h. For sterilization, scaffolds were soaked in 70%ethanol for 2 h, after which the ethanol solution was removed and thescaffolds were washed twice with 200 μL portions of D-PBS(−) buffer(Wako, cat. no. 045−29795). Finally, the scaffolds were incubated in200 μL of PBS buffer for 18 h in a sterile hood under sterilizing UVlight. hMSC were cultured in 75 cm2 polystyrene flasks using hMSCExpansion Media (StemXVivo), designed specifically for humanmesenchymal stem cells. Cultures contained 7−10 mL of hMSCExpansion Media and where incubated at 37 °C with 5% CO2 for 48 h.hMSC Expansion Media was replaced with a fresh portion after 24 h,at this time also containing the antibiotic-antimycotic Anti-Anti (LifeTechnologies, cat. no. 15240−062). Once the cultures reached 80−90% confluence, the cells were removed from the flasks by trypsindigestion and counted using a hemocytometer. To each wellcontaining a scaffold, 8000 cells in 200 μL of hMSC expansionmedia were added, and incubated at 37 °C with 5% CO2 for 24 h toallow for cell attachment. After 24 h, the medium was exchanged witha fresh 200 μL portion of hMSC expansion media containingantibiotic-antimycotic, and the cells were allowed to grow for anadditional 24 h. Viability was measured by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)(MTS) Assay (Promega), where at 48 h after hMSC seeding, 20 μL ofthe MTS reagent was added to each well containing a scaffold, andincubated at 37 °C with 5% CO2 for 1 h. The supernatant wascollected, and its absorbance measured at 490 nm using a MolecularDevices SpectraMax M3 plate reader. Each sample consisted of 3−5replicates. Viability was also observed by Invitrogen’s Live/Dead assay(Life Technologies). After the 48 h incubation, the medium wasremoved from the scaffolds, and 100 μL of a solution containingcalcein AM (2 mM) and ethidium homodimer-1 (4 mM) was added.After 30 min at 37 °C with 5% CO2, the scaffolds were removed fromthe 96-well plate, placed on a glass microscope slide, and viewed usinga ZeissLSM 700 confocal microscope. Excitation wavelengths of 488/555 nm were used to observe the fluorescence of live (green) and dead(red) cells.

Scanning Electron Microscopy. PHBU scaffolds were analyzedusing a JCM-6000 NeoScope Benchtop scanning electron microscopeset to backscattered electron image (BEI) at 10 kV acceleratingvoltage, high vacuum, and high probe current. To observe hMSCadhered to the scaffolds, we fixed samples from the Live/Dead assaywith a 3% glutaraldehyde solution for 4 h, followed by dehydrationusing a series of aqueous ethanol solutions of 70, 80, 90, 95, and99.5%. Each round of ethanol dehydration was performed for 1 h at 25°C, at which point the solution was completely replaced by the nexthigher concentration in the series. Following the final dehydrationstep, scaffolds were dried under vacuum for 24 h. Preparation formicroscopy involved mounting samples on carbon tape and sputtercoating for 30 s with gold.

Analysis of Mechanical Properties. Tensile strength wasmeasured using a Shimadzu EZ-LX HS universal tester equippedwith a 500 N load cell. PHBU samples with dimensions averaging 20 ×2 × 0.1 mm were pulled at a constant rate of 2 mm/min until break.The tensile strength, Young’s modulus, and elongation to break weredetermined on the basis of the generated stress/strain curves.

■ RESULTS

Production and Characterization of PHBU Copolymer.A PHA copolymer was designed with unsaturated repeating

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unit content that could be cross-linked to improve themechanical properties of the material to match those of softtissues such as cartilage. This material requires a high amount ofstiffness and enough flexibility to prevent brittleness. On thebasis of a previous study,28 a PHBU copolymer with 8% MCLcontent and 92% SCL content was produced for its balancedphysical properties and its ample amount of MCL content forcross-linking. The fatty acids chosen for this polymer werebutyric acid (cultured as sodium butyrate to reduce toxicity)and 10-undecenoic acid, which resulted in the copolymerpoly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxy-10-undecenoate](PHBU). A series of PHBU copolymers were produced with arange of MCL repeating unit contents in order to determinethe parameters to produce copolymers with a final desired ratioof repeating units. A standard curve depicting the ratio of fattyacid concentration to repeating unit composition for PHBUwas generated (Figure 1a). These results allowed for the designof a feeding strategy to produce PHA with 8% MCL content byadjusting the fatty acid ratio in the culture medium to thecorrect specifications based on the established trend. Therepeating unit content of PHBU was determined by 1HNMRspectroscopy (Figure 1b), where the ratio of the alkene protonsignal at 5.8 ppm to the polymer backbone stereocenter at 5.3ppm was taken as the MCL portion of the copolymer. Theyield of PHBU was approximately 50 wt % of the cell dryweight, which was similar to previous studies.28,29 The number-average molecular weight (Mn) of PHBU was estimated to be74.3 kDa by gel permeation chromatography (Table 1), andthis number was used in the stoichiometric calculations for thethiol-ene click reaction.Fabrication of PHBU scaffolds. Porous PHBU scaffolds

were produced with both small pore sizes of 30−50 μm andlarge pore sizes of 200−400 μm (Figure 2). The smaller poresresulted from the thermally induced phase separation of PHBUwith 1,4-dioxane, and the larger pores were derived from thesalt-leaching of sodium chloride. It was anticipated that thesmaller pore size would enhance cellular attachment andnutrient transport, whereas the larger pore size could promotevascularization of the scaffold if necessary. Analysis by scanningelectron microscopy (SEM) revealed a uniform dispersion ofpore sizes, with the walls of large pores containing the smallerpore size. This architecture should benefit transport of nutrientsand waste materials, as the large pores were interconnected by anetwork of smaller pores present throughout the scaffold.

These scaffolds were soft to the touch and spongelike, withmuch greater flexibility than the PHBU films of the samerepeating unit composition.

Gel Fraction of PHBU Scaffolds. After scaffold formation,the extent of polymer participation in the cross-linked network(gel fraction) was determined by swelling the material inchloroform (Scheme 1). In scaffolds that contained a 1:4 ratioof PETMP to alkene functional groups, the gel fraction wasdetermined to be 98%. This ratio was chosen in order tomaximize the amount of cross-linking in the scaffold, because ofthe four available thiol groups in PETMP. Scaffolds were alsoprepared with half the amount of PETMP cross-linker, and thisresulted in scaffolds with a gel fraction averaging 50%.

FT-IR Analysis of Cross-Linked PHBU. To assess thedegree of conversion to the cross-linked product, we analyzedPHBU and cross-linked PHBU materials by FT-IR (Figure 3).This qualitative measure of alkene content in the final polymershowed that the signal corresponding to the alkene functionalgroup in the side-chain of PHBU was reduced after the cross-linking reaction. Samples containing an equimolar concen-tration of PETMP thiol groups to PHBU alkenes (PHBUX98%)showed a reduction in the 1641 cm−1 signal by more than 90%,whereas samples containing half that concentration of PETMP(PHBUX50%) had a reduction in the alkene signal ofapproximately 50%. This result confirms that the concentrationof cross-linking can be controlled by the precise addition of thePETMP cross-linking agent.

Viability of Human Mesenchymal Stem Cells (hMSC)on PHBU Scaffolds. After the scaffolds were fabricated andsterilized, each one was seeded with hMSC and incubated for a

Figure 1. Analysis of produced poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxy-10-undecenoate] (PHBU). (A) Standard curve of PHBU. The 10-undecenoate content in the copolymer was monitored relative to the amount of 10-undecenoic acid present in the culture medium. The 10-undecenoate content in the polymer ranged from 5 to 30% with medium 10-undecenoate compositions ranging from 2 to 50%. The trend ofunsaturated fatty acid uptake was nonlinear, with less than incorporation after 12% PHU content. (B) 1HNMR of PHBU copolymer with 7% PHUcontent. Composition was determined using the ratio of the alkene proton signal at 5.8 ppm to the polymer backbone stereocenter proton at 5.3ppm.

Table 1. Molecular Weight and Yield of PHBU Samples

PHBUa Mn Mw Mw/Mn yield (% CDW)

5:95 71.7 ± 4.0 208.4 ± 7.3 2.9 ± 0.1 43.4 ± 3.910:90 63.7 ± 2.1 188.4 ± 2.8 3.0 ± 0.1 43.8 ± 2.320:80 75.8 ± 2.8 209.1 ± 3.3 2.8 ± 0.1 N.D.25:75 67.2 ± 3.4 201.3 ± 5.0 3.0 ± 0.1 N.D.50:50 93.2 ± 2.0 227.1 ± 2.0 2.4 ± 0.1 N.D.

aRatio of 3-hydroxyundecenoyl to 3-hydroxybutyrl repeating units inthe polymer. Mn, apparent number-average molecular weight ascompared to polystyrene standard; Mw, apparent weight-averagemolecular weight as compared to polystyrene standard; Mw/Mn,polydispersity; CDW, cell dry weight; N.D., No data

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total of 48 h. Cell viability measured by live/dead assay usingconfocal fluorescence microscopy showed similar amounts oflive and dead cells on the cross-linked scaffolds as compared tonon-cross-linked scaffolds (Figure 4). Cell viability measured byMTS assay showed no significant difference in metabolicactivity between the positive control with no scaffold andPHBU scaffold (p = 0.82). No significant difference was alsoobserved when comparing the PHBU scaffold and cross-linkedPHBU scaffold (p = 0.54), (Figure 5). Analysis of the cell-seeded PHBU scaffolds by SEM (Figure 6) revealed thatgreater amounts of extracellular matrix formed on small pore

size in the 30−50 μm range, indicating that this pore sizepromoted higher amounts of hMSC attachment and growth.

Mechanical Strength Analysis of Cross-Linked PHBU.The process of cross-linking PHBU resulted in significantincreases to tensile strength. The increase of gel fraction incross-linked PHBU correlated to an increase in tensile strength.PHBU cross-linked to 50% gel fraction exhibited an increase intensile strength from 8.5 to 11.2 MPa, whereas PHBU cross-linked to 98% gel fraction showed an increase to 26.2 MPa. Thestrain to failure did not change significantly between the threesamples, though there was a decrease in Young’s modulus withincreased cross-link density (Table 2). This decrease inmodulus is likely the plasticizing effect of the PETMP cross-linking agent. The ester functional groups of PETMP make itsimilar in structure to known plasticizers for PHAs,42 such asfatty acids and triglycerides. Notably, the shape of the PHBUstress/strain curve (Figure 7a) differs from the cross-linkedPHBU stress/strain curve (Figure 7b), where the cross-linkedsample changed to closely resemble a J-shape stress/straincurve.

■ DISCUSSION

Tissue-engineering requires that the material used to fabricatethe scaffold closely match that of the target tissue. In additionto meeting physical requirements, the material must also bebiocompatible and biodegradable. PHAs are suited for tissue-engineering applications due to their inherent biocompatibility,biodegradability, and diverse set of physical properties availableamong SCL-PHA, MCL-PHA, and PHA copolymers. Althoughthese attributes are found in other biopolymers such as PLA

Figure 2. Scanning electron micrographs of porous PHA scaffolds. (A) Large pore sizes (>200 μm) are the result of salt leaching, whereas (B) smallpore sizes are fabricated using thermally induced phase separation. The combination of large and small pores should facilitate in-growth of cells andnutrient transport, respectively.

Scheme 1. Reaction Scheme of the Thiol-ene Cross-Linking of PHBU

Figure 3. FT-IR of PHBU and cross-linked PHBU polymers. Evidenceof successful cross-linking was seen in the reduction of the signal foralkene functional group at 1641 cm−1. Extent of modificationcorrelated to the concentration of cross-linking agent in the reaction.

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and PCL, PHAs have the advantage of chemical modification tofurther tune the characteristics of the polymer to the targettissue. This secondary level of control in physical propertiesallows for PHAs to be tuned for specific tissue replacements ina way that is not possible with other biopolymers.PHAs are typically limited to very soft tissue applications

because of their low tensile strength.43 The cross-linked PHBUscaffolds produced in this study have substantial increases intensile strength compared to other SCL/MCL PHAs, withmechanical properties similar to connective tissues such asfibrocartilage and ligaments (Table 3). In addition to changes inphysical properties, the shape of the stress/strain profile ofcross-linked PHBU now more closely resembles that ofbiological tissue. MCL PHAs have large deformations when astress close to their ultimate tensile strength is applied. Duringthis period of deformation, dramatic increases in elongation areseen with little or no increase in measured tensile strength.Contrary to this, cross-linked materials have a J-shape stress-strain curve,44,45 where during the period of high elongation,

there is a distinct increase in measured tensile strength. Thistype of profile was observed in the cross-linked PHBU stress/strain curve of Figure 7b, where increased strain was coupledwith increased tensile strength over the entire curve. This resultindicated that the cross-linked PHBU had deformationbehavior similar to biopolymers like collagen, where overallmaterial toughness arises from high elasticity at lowdeformation forces and high stiffness at high deformationforces.Scaffold fabrication is a critical factor in whether or not the

implant will facilitate attachment and growth of new cells. Toproduce a PHBU scaffold with the necessary porousarchitecture, two methods for pore production were utilized.It was anticipated that the smaller pore size, produced bythermally induced phase separation, would enhance cellularattachment and nutrient transport, while the larger pore sizefrom salt leaching could promote vascularization of the scaffoldif necessary. Although there is agreement that high porosity isnecessary for nutrient transport and cell attachment,35 there areconflicting arguments on the size of pore necessary for the mostefficient cell adhesion to the scaffold. Certain studies haveshown success with scaffolds containing <50 μm pore sizes.10 Inthose experiments, human bone marrow mesenchymal stemcells were able to attach and proliferate on scaffolds producedfrom thermally induced phase separation, similar to the resultsof this study. However, other research has shown that poresizes of >100 μm are necessary for cell migration and transport,and pore sizes >300 μm are preferred for the growth ofcapillaries and new bone.6 These previous studies guided theapproach taken in our study where PHBU scaffolds with twodifferent pore sizes were produced: small pore sizes weregenerated through thermally induced phase separation of thepolymer in frozen 1,4-dioxane, and large pore sizes wereintroduced through a salt-leaching technique with sodiumchloride. The benefit of the scaffold fabrication methoddescribed here is the ability to alter pore sizes as necessary

Figure 4. Fluorescence microscopy of human mesenchymal stem cells (hMSC) grown on cross-linked PHBU scaffolds for 48 h. Live cells werestained with calcein AM, a membrane permeable dye that fluoresces green when exposed to intracellular esterases, and dead cells weresimultaneously stained with ethidium homodimer-1, a membrane impermeable die that fluoresces red when bound to nucleic acids. (A) Cells seededon PHBU scaffold. (B) Cells seeded on PHBUX98% scaffold. Cell viability was unaffected by cross-linking.

Figure 5. MTS viability assay of hMSC grown on PHBUX50% scaffold.The differences between the positive control and PHBUX50% scaffolds,and between the PHBU and PHBUX50% scaffolds were statisticallyinsignificant as determined by unpaired t test values of p = 0.82 and p= 0.54, respectively.

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for the desired application with minor procedural changes.Tissue-engineering scaffolds that require larger vasculature orbone formation need only exchange the sodium chloridecrystals for a larger porogen. Based on the results shown here,scaffolds with small 30 μm pores promoted higher extracellularmatrix formation, which may be due to better cell adhesion.The reduced ECM formation observed on PHBU scaffolds withonly large pores (>300 μm) is likely due to surface topologyand not nutrient transport, because of the localization of thecells to the surface during the short incubation period.Cross-linking the PHBU scaffold resulted in a dramatic

increase in tensile strength. With tensile strengths as high as26.2 MPa, this material may find use as cartilage or otherconnective tissue substitute.3,46 Interestingly, the cross-linkedscaffolds were able to maintain their geometry better than thenon-cross-linked scaffolds after incubation for 48 h in hMSCexpansion media at 37 °C. When physically transferring the

cell-seeded scaffolds for microscopy, non-cross-linked PHBUscaffolds were easily damaged despite gentle handling, whereasthe cross-linked PHBU scaffolds had higher durability andeasily maintained their shape even with rough handling. Cross-linking drastically improved the performance of the scaffold inan aqueous environment, which is a notable drawback to otherbiopolymers used as tissue-engineering materials.47 Thisaqueous stability may become important for maintaining thestrength of the scaffold after extended exposure in vivo.An important point of consideration is the effect of cross-

linking on the rate of biodegradation. It is known that cross-linking can affect the rate of polymer biodegradation,46−49

however these differences vary drastically depending on thetype of polymer and degree of cross-linking. In these studies,biodegradation took up to twice as long (16 vs 8 days) in highlycross-linked samples. Further study into the biodegradation ofcross-linked PHAs would be necessary to determine whethertheir rates are suitable for tissue-engineering scaffolds.50

This study has shown that thiol-ene click chemistry can beused to efficiently cross-link unsaturated PHA polymers inorder to dramatically improve the strength of the materials. Itwas found that the chemical cross-linking of the PHA scaffoldsdid not result in significant cytotoxicity toward humanmesenchymal stem cells. Although it is encouraging that thenew chemical cross-links are not cytotoxic, further tests are

Figure 6. Scanning electron micrographs of human mesenchymal stem cells grown on and then fixed to cross-linked PHA scaffolds with (A, B) large(>200 μm) and (C, D) small (30 μm) pore size. Greater amounts of extracellular matrix material was observed on regions of scaffold consistingmainly of small pore size.

Table 2. Physical Properties of Cross-Linked PHBU

sample modulus (MPa) stress (MPa) strain (%)

PHBU 367.5 ± 43.8 8.5 ± 0.5 88.8 ± 19.2PHBUX50%

a 258.3 ± 67.9 11.2 ± 0.2 120.3 ± 16.5PHBUX98%

b 205.5 ± 58.7 26.2 ± 1.1 95.2 ± 8.3a50% gel fraction. b98% gel fraction; modulus, Young’s modulus;stress, tensile strength; strain, elongation at break; MPa, megapascals.

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necessary to conclude that the cross-linked material remainsbiocompatible. In future in vivo studies, biocompatibility couldbe assessed by implanting the cross-linked PHA into mice andmonitoring inflammation.51

Thiol-ene click chemistry is not limited to cross-linkingreactions. This chemistry may also be employed to incorporatenew functional groups into the side chain of the polymer,leading to control over other properties such as polarity,solubility, processing temperatures, crystallinity, and biodegrad-ability dependent on the modification. By controlling therepeating unit composition of the PHA polymers produced byE. coli LSBJ, we can now approach a two-dimensional designframework where the polymer’s properties can be defined bythe ratio of SCL to MCL repeating units in addition to the newchemical moieties attached to the side chains resulting in amatrix of tunable properties.

■ CONCLUSIONS

The three objectives of this work were to (1) produce anunsaturated PHA that could be cross-linked by thiol-ene clickchemistry, (2) determine the change in the physical propertiesof PHA based on cross-link density, and (3) assess the viabilityof human cell cultures in the presence of the chemicallymodified PHA. This work was successful in producing a SCL/MCL PHA copolymer with tunable repeating unit compositionfor chemical modification. Using thiol-ene click chemistry,cross-linked PHBU samples were produced with varying cross-link density based on the concentration of the cross-linkingreagent. The change in physical properties of the highly cross-linked PHBU to 26.2 MPa tensile strength and 205.5−280.5MPa Young’s modulus will allow for new soft tissueapplications unavailable to unmodified PHBU. When culturedwith hMSC, PHBU and cross-linked PHBU did not have asignificant impact on cell viability. For use in tissue engineering,it was critical that the chemical modification by thiol-ene clickchemistry not induce cytotoxicity, which would have renderedthe scaffolds useless for regenerative medicine applications.This study represents a critical first step in the use of chemicallymodified PHA in tissue-engineering scaffolds. The lack ofcytotoxicity toward hMSC will allow for future experiments toassess the in vivo biocompatibility and biodegradation ofchemically cross-linked PHA. The three objectives of this workwere accomplished with the production of an unsaturated PHAcopolymer with improved physical properties through chemicalcross-linking that did not adversely affect cellular viability fortissue engineering applications.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: ctnomura@esf.edu. Telephone: 315-470-6854. Fax:315-470-6856.

Author ContributionsThe manuscript was written by A.C.L. and C.T.N. Experimentswere designed by A.C.L., C.T.N., and K.N. A.S. and F.F.T.assisted in PHA production. A.C.L. carried out all otherexperiments.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors acknowledge support from NSF CBET 1263905awarded to C.T.N. and an award to A.C.L. from the NSF EastAsia Pacific Summer Institutes (EAPSI) program in collabo-ration with the Japanese Society for the Promotion of Science(JSPS). Special thanks go to Keiji Numata, who was willing tohost this research project in his lab at the RIKEN. We alsothank Jo-Ann Chuah and Jose Manuel Ageitos for theirassistance with hMSC cultures and microscopy. F. F. Twigg wassupported by NSF REU 1156942 awarded to J. M.Hasenwinkel and P. T. Mather of the Syracuse BiomaterialsInstitute.

■ REFERENCES(1) Chan, B. P.; Leong, K. W. Scaffolding in tissue engineering:General approaches and tissue-specific considerations. Eur. Spine J.2008, 17, 467−479.(2) Haugh, M. G.; Murphy, C. M.; Mckiernan, R. C.; Altenbuchner,C.; O’ Brien, F. J. Cell Attachment, Proliferation, and Migration

Figure 7. Stress−strain curves of (A) PHBU and B) PHBUX98%. Anincrease in tensile strength from 8.5 MPa in the non-cross-linkedPHBU to 26.2 MPa in the cross-linked PHBU was observed. Theshape of the stress strain curve became nearly J-shaped in the cross-linked polymer, where increasing strain on the sample resulted incontinued increase in observed stress.

Table 3. Comparative Properties of Natural and SyntheticPolymers

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PHBU 367.5 8.5 88.8 this studya

PHBUX98% 205.5 26.2 95.2 this studypolylactic acid 1200 28 6 41polycaprolactone 400 16 80 41skin 0.1−0.2 7.6 60−80 3,39fibrocartilage 159.1 27.5 3,38ligaments 303.0 29.5 3low density polyethylene 200 10 620 25polytetrafluoroethylene 500 27.5 3silicone rubber 8 7.6 3aPolymer generated from 10-undecenoic acid and sodium butyrate byE. coli LSBJ.

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