Research Article Synthesis and Characterization of Biodegradable...

Research ArticleSynthesis and Characterization of Biodegradable Polyurethanefor Hypopharyngeal Tissue Engineering

Zhisen Shen1 Dakai Lu2 Qun Li2 Zongyong Zhang3 and Yabin Zhu2

1Lihuili Hospital Ningbo University 818 Fenghua Road Ningbo 315211 China2The Medical School Ningbo University 818 Fenghua Road Ningbo 315211 China3School of Chemical Engineering Ningbo University of Technology 201 Fenghua Road Ningbo 315211 China

Correspondence should be addressed to Zhisen Shen szs7216163com and Yabin Zhu zhuyabinnbueducn

Received 3 November 2014 Revised 18 January 2015 Accepted 13 February 2015

Academic Editor Esmaiel Jabbari

Copyright copy 2015 Zhisen Shen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Biodegradable crosslinked polyurethane (cPU)was synthesized using polyethylene glycol (PEG) L-lactide (L-LA) and hexamethy-lene diisocyanate (HDI) with iron acetylacetonate (Fe(acac)

3) as the catalyst and PEG as the extender Chemical components of the

obtained polymers were characterized by FTIR spectroscopy 1H NMR spectra and Gel Permeation Chromatography (GPC) Thethermodynamic properties mechanical behaviors surface hydrophilicity degradability and cytotoxicity were tested via differentialscanning calorimetry (DSC) tensile tests contact angle measurements and cell culture The results show that the synthesized cPUpossessed good flexibility with quite low glass transition temperature (119879

119892 minus22∘C) and good wettability Water uptake measured as

high as 2297plusmn 187These propertiesmake cPU a good candidatematerial for engineering soft tissues such as the hypopharynx Invitro and in vivo tests showed that cPU has the ability to support the growth of human hypopharyngeal fibroblasts and angiogenesiswas observed around cPU after it was implanted subcutaneously in SD rats

1 Introduction

Recently the rate of pharyngeal cancer has been increasingyear after year due to alcohol and tobacco abuse [1] Inthe literature 60ndash80 of hypopharyngeal head and necktumor patients were reported to have developed ipsilaterallymph node metastasis upon presentation of clinical symp-toms leading an overall five-year survival rate of 334[2 3] Surgical removal of the apparent tumor is still animportant clinical treatment although evidence suggeststhat chemotherapy and radiotherapy can help to controltumor growth and promote patient survival rates [4] Tumorresection results in tissue defects which inevitably leadsto chronic health problems including hindered breathingswallowing vocalizing and possible mental illness Clinicalsurgery is usually performed using jejunal free flap lateralthigh flap or pectoralis major myocutaneous flap as thesubstitutes However due to the complex structure of thethroat these substitutes cannot restore pharyngeal functionand often result in flap necrosis infection fistula or stenosis

[5ndash8] Therefore it is necessary to find effective and safesubstitutes for laryngeal reconstruction

The rapid development of tissue engineering brings newpossibilities for laryngeal and hypopharyngeal reconstruc-tion Biodegradablematerials with goodmechanical strengthelasticity and nontoxicity are good potential candidates asbiological substitutes Poly(lactic acid) (PLA) is a promisingmaterial which has been approved by the Food and DrugAdministration (FDA) for medical applications Howeverthere are some drawbacks with the use of PLA as a matrixfor hypopharyngeal tissue For example it is a stiff polymerwith a high glass transition temperature (119879

119892) of 65∘C which

does not meet the requirements of laryngeal tissue growthPolyurethane is a widely used material in tissue engi-

neering because of its good mechanical properties biocom-patibility and biodegradability [9 10] It is usually synthe-sized via a crosslinking reaction between diisocyanate andpolyhydric hydroxy with polyol or diamine as the extenderSome components such as caprolactone poly(ethyleneoxide)lactic acid and ricinoleic acid have been experimented

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 871202 11 pageshttpdxdoiorg1011552015871202

2 BioMed Research International

Table 1 The component feeding ratios and GPC measurements of prepolymer PLEG

Copolymer PEGL-LA in feeding (mol) 119872119899(Da) 119872

119908(Da) 119872

119908119872119899

PLEG 1 12 1 4365 5926 13576PLEG 2 1 1 4702 5977 12709PLEG 3 1 2 5422 6311 11638PLEG 4 1 5 6046 7312 12090

with in the production of thermoplastic and biodegradablepolyurethanes [11ndash14] In our previous work we studied thecatalysis of aliphatic ester polymerization by low toxicityiron compounds instead of the stannum compounds usedcommonly in research orand industry [15] Using thisapproach we synthesized a biocompatible and biodegradablecopolymer with 119879

119892of 56∘C from monomers of L-lactide

poly(ethylene glycol) (PEG) and NIPAAm [16] Howeverthe previously published polymer was too rigid to be usedin reconstruction of soft tissues like the hypopharynx Inthis study we have produced a material with lower 119879

119892

better strength and superior biodegradability using reactionsbetween L-lactide PEG and hexamethylene diisocyanate(HDI) The frequency and chemistry of the reaction werecharacterized by Fourier transform infrared spectroscopy(FTIR) Hydrogen-1 nuclear magnetic resonance (1H NMR)spectra and Gel Permeation Chromatography (GPC) Thenovel materialrsquos mechanical property hydrophilicity degrad-ability cytocompatibility and in vivo biocompatibility wereevaluated We believe that the material presented in thispaper will be a good substitute for hypopharyngeal tissueengineering

2 Materials and Methods

21 Materials PEG (Mn 2000) dichloromethane (CH2Cl2)

ethanol and ethyl acetate were purchased from SinopharmChemical Reagent Co Ltd L-lactide (L-LA) was supplied byJinan Daigang Biological Material Company It was recrystal-lized three times using ethyl acetate as the solvent prior toreaction Iron (III) acetylacetonate (Fe(acac)

3) and HDI were

purchased from Aladdin Reagent Co Ltd ChinaTrypsin (1 250 GNM) was purchased from Beijing

Genosys Scientific Co China Mouse anti-vimentin andFITC-conjugated goat anti-mouse IgG were from WuhanBoster Bio-Engineering Co Ltd IRDye 680RD goat anti-mouse IgG (H+L) was purchased from LI-COR BiosciencesUSA All cell culture reagents were purchased from HyCloneunless otherwise specified All chemical reagents used forWestern blotting were purchased from Beyotime Institute ofBiotech Jiangsu China Phosphate buffer saline (PBS pH74)used in cell culture was sterilized prior to use Sprague-Dawley (SD) male rats were obtained from the ExperimentalAnimal Center of Ningbo University

22 Synthesis of PLA-PEG-PLA Prepolymer (PLEG) Theprepolymer PLEGwas synthesized using L-LA and PEG (Mn2000) as monomers and Fe(acac)

3as the catalyst as per our

previous protocol [12] Briefly predetermined amounts of

PEG and L-LA were added into a polymerization tube withFe(acac)

3(05 weight ratio to total reactants resolved in

CH2Cl2) as the catalyst After degassing for 2 h at 60∘C the

tube was sealed under vacuum and placed entirely into an oilbath and the bath temperature was maintained at 130∘C for20 h with stirring The polymerization tube was then takenout of the oil bath and cooled to room temperature Tenmilliliters CH

2Cl2was added to dissolve the product After

precipitation in ethanol at 0∘C the three-block PLA-PEG-PLA prepolymer (PLEG) with different ratios of monomerswas obtained (Table 1) The PLEG was subsequently dried ina vacuum oven and kept in the dryer for the next reaction

23 Preparation of Crosslinked Polyurethane PrepolymerPLEG PEG and HDI (molar ratio 165 10 29) were mixedand heated to 80∘C for 8 h under nitrogen protection toallow crosslinking to occur PLEG and PEG were sepa-rately dehydrated via degassing for 1 h at 110∘C prior to thecrosslinking reaction A shallow yellow sticky solution wasformed at the end of the reaction It was dissolved in CH

2Cl2

to produce a 50 (v v) solution which was cast onto apolydimethylsiloxane (PDMS) film and dried in an oven at60∘C overnight to yield a flat approximately 10mm thickcPU membrane PDMS was used as the substrate as thesynthesized cPU membrane could easily be peeled off

24 Characterization of Prepolymer and Crosslinked Polymer

241 PLEGPrepolymer FTIR spectrawere recorded using anFTIR instrument (Digilab FTS 3100 USA) with 4 cmminus1 reso-lution and a measuring period of 500ndash4000 cmminus1 The reac-tion chemistry of prepolymer PLEG was confirmed via1H NMR measurement PLEG sample was dissolved inCDCl

3and tested on a NMR spectrometer (Bruker Avance

400MHz Switzerland) The molecular weight (Mn and Mw)and molecular weight distribution (MwMn) were deter-mined by GPC (Polymer Laboratories PL-GPC 50 plusEngland) using polystyrene as the standard The analysis wasperformed at 40∘C using tetrahydrofuran (THF) as the eluentat a flow rate of 10mLmin

242 Structure and Characteristics of cPU The thermalproperties of cPU were tested by differential scanningcalorimetry (DSC PyrisDiamondUSA) under nitrogenThefirst heating was from 25 to 100∘C at a rate of 20∘Cmin with1min station to clear the thermal history then the samplewas cooled down to minus50∘C at a rate of 10∘Cmin The secondheating started from minus50∘C and increased to 100∘C at a rate of20∘Cmin The glass transition temperature (119879

119892) and melting

BioMed Research International 3

point (119879119898) values were taken from the second round of

heatingThe static contact angles of cPU were surveyed on

a surface tension-contact angle meter (DIGIDROP GBXFrance) and the droplet dynamic contact angle was testedon Dataphysics OCA20 (Germany) at ambient humidity andtemperature A drop of deionized water was approximately10 120583L in volume The contact angle values of samples wereaveraged from three different locations and expressed asmean plusmn standard deviation (SD)

Mechanical properties of cPU were tested with a tensiletester (Instron 3366 USA) using a linear deformation rateof 10mmmin at 25∘C Dumbell-shaped polyurethane mem-branes with a gauge length of 30mm and cross-sectional areaof 02ndash03mm times 1mmwere used Larynx tissue was obtainedfrom Lihuili hospital (Ningbo China) under agreementof laryngeal carcinoma patients It was tested and used asreferences to compare between the synthesized materials andthe native tissue Three repeats were performed for eachsample

Water uptakewas tested and calculated as follows119877() =[(1198821minus 1198820)1198820] times 100 where119882

0was the original weight

and1198821was the weight of cPU after being dipped in deionized

water for 10 h at room temperature Three repeats wereperformed for cPU

25 Degradation Test Samples were cut into pieces 40 times40 times 1mm in size (initial weight 119882

0) and incubated for

1 5 10 20 40 or 80 d in PBS (pH = 74) supplementedwith 100UmL penicillin-streptomycin at 37∘C After rinsingin water and drying in a vacuum oven samples weights(119882119897) were measured on an electronic analytical balance

(Sartorius BS 224s Germany) The percentage of weight losswas calculated using the following formula

Weight loss () =1198820minus119882119897

1198820

times 100 (1)

1198820represents the initial weight of the sample (mg) and

119882119897represents the measured weight (mg) of the same sample

after different degradation times The degradation tests wereperformed in triplicate for each time point

26 Primary Human Hypopharyngeal Fibroblast CultureHuman fibroblasts were obtained from patient hypopharynxconnective tissue (sample from Lihuili Hospital in NingboChina under agreement of a pharyngeal carcinoma patient)The tissue sample was rinsed well in sterile PBS containingantibiotics (1000UmL penicillin and 1000 120583gmL strepto-mycin sulfate) and sterilized in 75 ethanol for 5 s and in1000 ppm NaClO solution with PBS washing between stepsAfter that the tissue was cut into cubes which were approx-imately 1 times 1 times 1mm in dimension and attached to a cultureflask (Corning USA) containing a small amount of culturemedium Dulbeccorsquos Modified Eaglersquos Medium (DMEM)fetal bovine serum (FBS 10) penicillin (50 IUmL) andstreptomycin (50 IUmL)The culture mediumwas amendedto cover the tissue at the next day After several daysfibroblasts extended from the tissue cubes and attached to

the culture plateThese primary fibroblasts were collected andsubcultured for passaging

The cells with passage 2ndash4were seeded on cPUmembraneat the density of 4 times 104 cellsmL cPU membrane was cutand prelaid on culture wells (96-well culture plate) with tissueculture polystyrene (TCPS) (Corning USA) as the positivereference The culture was incubated in medium consistingof FBS (10 vv) and DMEM supplemented with penicillin(50 IUmL) and streptomycin (50 IUmL) in a humidified airof 5 CO

2at 37∘C The culture medium was changed every

2 d After incubation for some time cells were fixed with 25glutaraldehyde for HampE and immunofluorescent staining

27 Hematoxylin and Eosin (HampE) Staining HampE stainingwas used to help better visualize the density and morphologyof fibroblasts on the cPU membrane due to the opacity ofthe membrane The cultures were rinsed three times withPBS and fixed with 4 paraformaldehyde (Sigma USA) for30 minutes at room temperature The cultures were rinsedthrice with water to remove residual paraformaldehyde andphosphate salts Subsequently they were immersed in hema-toxylin solution (Beijing Solarbio Science amp TechnologyChina) for 30min to stain cell nuclei followed by dippingin 1 hydrochloric acidalcohol solution for 30 s to removethe excessive hematoxylin and washing with running waterfor 5min Finally the samples were immersed in 05 eosinsolution (Beijing Solarbio Science amp Technology China) for1min to stain the cytoplasmHampE staining images were takenunder light microscopy (Model CX40 Olympus Japan)

28 Immunofluorescent Staining The fibroblasts seeded oncPU membranes were fixed in 4 paraformaldehyde for10min at room temperature washed with PBS thrice for5min each time soaked in 02 Triton X-100 (BeijingSolarbio ScienceampTechnology China) for 20min and rinsedin PBS thrice for 10min each time After that the sampleswere blocked in 10 goat serumPBS for 20min at 37∘Cfollowed by incubation in the mouse anti-vimentin primaryantibody (1 200 dilution in PBS) at 4∘C overnight Afterrinsing with PBS the samples were incubated in FITC-conjugated goat anti-mouse IgG (1 50 dilution in PBS) for2 h at 37∘C in the dark room Finally after washing with PBSthe samples were dipped in a 46-diamidino-2-phenylindoledihydrochloride (DAPI)PBS solution (Sigma 3mgmL) for5min to stain the nuclei (blue fluorescence) The cells wereobserved under confocal laser scanning microscopy (CLSMOlympus Fluoview-1000)

29 Mitochondrial Activity Assay Mitochondrial activity ofthe cells was measured using the MTT method at days 15 10 and 14 respectively Twenty microliters of 05mgmLmethylthiazolyldiphenyl-tetrazolium bromide (MTT) solu-tion (05mgmL Beijing Solarbio Science amp TechnologyChina) was added to each well of the 96-well plate culturesand incubated at 37∘C for 4 h in the dark 150mL dimethyl-sulphoxide (DMSO) was subsequently added to each cultureto dissolve the purple formazan crystal The absorbance (ODat 490 nm) was recorded with an ELISA reader (MaxM5


Spectra USA) The absorbance of the same material in thesame solution containing no cells was used as blank referenceCells cultured on the tissue culture polystyrene (TCPS) wereused as the positive reference Triplicates of each sample wereaveraged

210 Western Blotting Cells grown on cPU membrane andTCPS 24-well culture plates for 14 d were washed three timeswith PBS for 5min each time Two hundred microlitersof radioimmunoprecipitation assay lysis buffer (RIPA) con-taining phenylmethylsulfonyl fluoride (PMSF) (RIPA PMSF100 1 v v) (Membrane and Cytosol Protein Extraction KitBeijing Solarbio Science amp Technology China) was addedto each well and kept for 30min on ice The cell lysatewas collected and centrifuged at 12000 rpm for 5min at4∘CThe supernatant was collected in a new microcentrifugetube Twenty-five microliters of the supernatant was mixedwith 5 120583L 5X loading buffer and loaded onto a 12 sodiumdodecyl sulfate (SDS) polyacrylamide gel Electrophoresiswas performed in running buffer at 100V for 2 h The sep-arated proteins were then transferred onto a polyvinylidenefluoride membrane (PVDF Roche Diagnostics) at 70V for2 h After blocking with Tris-buffered saline (TBS) containing5 skim milk for 1 h at room temperature the membranewas incubated in anti-vimentin mouse monoclonal antibody(1 500 dilution in BSA blocking solution) overnight at 4∘CAfter three rinses with 005 Tween-20 in TBS (vv) (TBS-T) the PVDF membrane was incubated in goat IR Dye 680anti-mouse IgG (H+L) (1 15000 dilution in TBS-T) for 2 h at37∘C The membrane was then scanned and analyzed usingthe Odyssey infrared scanning imaging system (Odyssey LI-COR USA) The gradation of the target band was calculatedwith the Odyssey infrared scanning imaging system forstatistical analysis Beta-actin was used to normalize thecellular protein content The results presented were fromthree separate experiments

211 In Vivo Biocompatibility In order to assay the mate-rialrsquos in vivo biocompatibility female SD rats (3 monthsold 250ndash300 g) were anesthetized with 5 chloral hydrate(intraperitoneal injection 6mgkg) and implanted subcuta-neously with sterilized cPU discs (7mm in diameter) After apredetermined period of time the SD rats were sacrificed andsamples were explanted with a small amount of surroundingtissue These tissue specimens were fixed in 10 formalinfor 1 h followed by freeze-embedding and microtome slicinginto 4 120583m sections Samples were stained with HampE stainand were analyzed under light microscopy (Olympus CX40Japan) Images were captured by digital camera (PL-B623CUPixelink Canada)

The animals used in this study were treated in accordancewith the ethical committee of Ningbo University and theNIHrsquos Principles of Laboratory Animal Care

3 Results and Discussion

31 Characterization of PLEG Prepolymer The PLEG pre-polymer was synthesized from the reaction of L-LA and PEG

(molecular weight 2000 Dalton) using a low toxicity ironcompound as the catalyst (Scheme 1(a) [16]) The effect ofvarious ratios of L-LA and PEG on the productrsquos molecularweight was explored (Table 1)Themolecular weight of PLEGincreased with increasing quantities of L-LA The molecularweight distribution 116ndash135 is very narrow which showedthe low polydispersity of all products due to effective poly-merization The chemistry of PLEG was verified by FTIRspectra (Figure 1) which showed characteristic spectra forPEG (b) L-LA (c) andPLEG4 (d) Curve (d) (PLEG) appearsto be a sum of curve (b) (PEG) and curve (c) (monomeric L-LA) with a specific peak at 1756 cmminus1 which can be attributedto ester stretching absorption of L-LA and PLEG The widepeak at 3450 cmminus1 can be attributed to hydroxyl groups inthe end group of PEG and PLEG (this peak is not presentin the L-LA curve) These FTIR characteristics suggest thatpolymerization occurred between PEG and L-LA to yield thethree-block polymer PLEG

The reaction was also analyzed by 1HNMRusing PLEG 4(mole ratio of PEG L-LA = 1 5) as the experimental sample(Figure 2) Particularly both protons of the methyl CH

3at

157 ppm (a) and of theCHwith its tertiary carbon attached tohydroxyl and carbonyl groups at 517 ppm (c) from the L-LAmonomer showed the polylactide (PLA) block while protonsof the CH

2CH2segment attached to two oxygen atoms at

364 ppm (b) were determined to originate from the PEGmoiety The peak area ratio of (a) to (c) was approximately3 1 and confirmed the block structure of -O(CH

3)CHCO-

in PLEG The peak ratio of (b) to ((a)+(c)) 71 416 isclose to 2 1 demonstrating that the chains of two PLAsubblocks were similar that is 119909 asymp 119910 in Scheme 1(a) Theseresults indicate that two average blocks of homopolymerPLA and one block of PEG formed the prepolymer PLEG4 with uniform block weight This result is considered tobe in agreement with the molecular weight measurementof PLEG 4 (6046Da Table 1) in which each PEG moietyweighs 2000Da and each PLA moiety weighs approximately2000Da

In order to reduce the crystallinity of the prepolymer andto obtain a crosslinked polyurethane with good flexibility(low 119879

119892) we designed a ring opening reaction between LLA

and PEG to produce lower molecular weight prepolymer byusing higher amounts of catalyst than those in our previouswork [16] Considering its uniform structure molecularweight and polydispersity PLEG 4 was selected as thereactant for downstream reactions with PEG and HDI

32 Chemistry and Properties of cPU The crosslinking ofPLEG 4 and HDI was performed with PEG (Mn 2000) asthe extender to yield the desired cPU (Scheme 1(b)) Thiscrosslinking reaction was confirmed by FTIR spectra (Fig-ure 1) which showed spectra for HDI (a) PEG (b) PLEG (d)and the product cPU (e) Curve (e) (cPU) showed the samepeaks as HDI (a) PEG (curve (b)) and PLEG (curve (d)) butwithout the peak at 2210 cmminus1 which is a specific peak of the-NCO- group from HDI which shows that there is no active-NCO- group in the product Peaks at 3320 and 1627 cmminus1 incPU were attributed to C=O stretching absorption in amide


H H

O

O

O

C

C

OOH

PEG

(b)

(a)

L-LA

OCH

O O

CH OH

PLA-PEG-PLA (PLEG)

OCCOCH2CH2 OCH2CH2n

x

n

y

+Fe(acac)3

135∘CCH3 CH3

PLA-PEG-PLA + OCN NCO + PEG Crosslink6

CH2CH2

simsimsimsim

simsimsimsim

simsimsimsim

simsimsimsim

O-OC-HN-(CH2)6-N-CO-O


CO-N

H-(CH

2 )6 -HN

-CO-

-

Scheme 1 Synthesis of oligomer PLA-PEG-PLA (P) using Fe(acac)3as the catalyst ((a) cited from [16]) and crosslinking of oligomer PLA-

PEG-PLA HDI and PEG (b)

Table 2 Properties of cPU film The cPU was prepared by crosslinking of PLEG4 PEG and HDI (mole ratio 165 10 29)

119879119892(∘C) 119879

119898(∘C) O (MPa) 120576 () 119864 (MPa) Static contact angle (∘) Water uptake ()

minus221 plusmn 24 615 plusmn 35 479 plusmn 076 725 plusmn 154 314 plusmn 28 711 plusmn 14 2297 plusmn 187

050

100150200250300350400450500

Tran

smitt

ance

2270

1756

3507 33202866 1750

16271262

1094

(a)

(b)

(c)

(d)

(e)

4000 3500 3000 2500 2000 1500 1000 500

Wave number (cmminus1)

minus100

minus50

Figure 1 FTIR spectra of HDI (a) PEG (b) L-LA (c) PLEG 4 (d)and cPU (e) The resolution is 4 cmminus1 and the measuring period is500ndash4000 cmminus1

groups (O=C-NRR1015840) while the peak at 3450 cmminus1 (-OH) wasweakened A peak at 3507 cmminus1 suggesting N-H stretchingappeared Peaks at 1750 cmminus1 (ester stretching absorption)2866 cmminus1 and 1094 cmminus1 (C-H and ether C-O-C stretchingabsorption originating from PEG) were also observed inthe product cPU Strong absorption from C-H stretching

H OCH CO

OC CH

x

n

y

OHO

(a)

(b)(c)

1 2 3 4 5 6(ppm)

(b) 364

PLEGCH3

CH3

OCH2CH2

(c) 517

(a) 157Δ316

Δ100

Δ710

Figure 2 1H NMR spectrum of prepolymer PLEG 4

suggested a large amount of PEG component All these resultstogether imply that crosslinking reactions took place betweenthe reactants HDI PEG and PLEG

The thermal and mechanical properties of cPU as well asits wettability and degradation were evaluated The curve inFigure 3(a) shows that the glass transition temperature (119879

119892)

and melting point (119879119898) occurred at minus221∘C and 615∘C This

quite low 119879119892displayed that cPU was very flexible at body

temperature 37∘C These thermal characteristics meet themechanical requirements for artificial materials to be used asbiomatrix for soft tissues


0 10 20 30 40 50 60 70 80

0

1

2

3

4

5

Stre

ss (M

Pa)

Strain ()

(a)

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Stre

ss (M

Pa)

Strain ()minus20

(b)

Figure 3 Stress-strain curve of the crosslinked cPU film (a) and larynx tissue (b)

40

0 50 100 150 200

45

50

55

60

65

70

75

Time (s)

Dyn

amic

cont

act a

ngle

(deg

)

(a)

40

30

20

10

0 20 40 60 80

50

60W

eigh

t los

s (

)

Time (d)

(b)

Figure 4 Dynamic contact angle (a) and weight loss (b) of cPU as a function of time The specimens were immersed in sterilized PBS (pH74) at 37∘C and kept in a sealed container during weight loss measurement

(a) (b)

Figure 5 Lightmicroscopy images of cells cultured on cPUmembrane for 2 d (a) and 14 d (b) Cells were stained byHampEThe seeding densitywas 4 times 104 cellsmL Scale bar 100 120583m


00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145

Figure 6Mitochondrial activity as shown by absorbance at 490 nmCells were seeded at a density of 4 times 104 cellsmL and cultured for 1day 5 days 10 days and 14 days

The mechanical properties of cPU are shown as a stressstrength-strain curve (Figure 3(a)) The maximum strengthand ultimate strain were recorded as 479 plusmn 076MPa and725 plusmn 154 Youngrsquos modulus was 314 plusmn 28MPa (Table 2)which was stronger than natural materials such as collagen(031MPa) [17] or chitosan (055MPa) [18] and biodegradablepolymers such as polyvinyl pyrrolidone (035MPa) [19] oraloe vera (15MPa) [20] both of which are often used inbiomedical applications However it is much weaker thanthe industry poly(ester urethane) (58213 NAT 022) whosemaximum strength and ultimate strain are 17MPa and 900respectively [21] Moreover the synthesized cPU in ourcurrent study is somewhat brittle as its ultimate strain is725 plusmn 154 After that the material broke stone-droppedlyIn order to know the mechanics of natural tissues larynxwas tested on the same system with the same parame-ters Its maximum strength and ultimate strain was 965 plusmn024MPa and 142 plusmn 131 respectively Youngrsquos moduluswas 6928 plusmn 321MPa (Figure 3(b)) The nature of the larynxseems stronger and more rigid than our synthesized cPUHypopharynx was supposed to be softer and more flexiblethan larynx However we are short of the detail data due toshortage of the tissue at present

An ideal material for tissue engineering should possessgood hydrophilicity and biocompatibilityThus a hydrophilicPEG component was introduced PEG is composed ofrepeating oxyethylene groups leading to good hydrophilicityand lipophilicity It is the most common material used toimprove hydrophilicity in a scaffold and further promotesthe biocompatability and cytocompatibility of the scaffold[22 23] In our case PEG was introduced as a component ofour materialThe hydrophilicity of the cPU was evaluated viastatic and dynamic contact angle measurements The instant

water contact angle was measured as 711 plusmn 14∘ (Table 2)This value decreased rapidly with time (Figure 4(a)) After200 s the water drop disappeared presumably absorbedby the material We also measured the maximum wateruptake 2297 plusmn 187 (Table 2) after dipping the materialin water for 10 h at which point it became a hydrogel withgood flexibility and softness which are good characteristicsfor hypopharyngeal tissue engineering An important con-sideration when designing scaffolds for tissue engineeringis degradation as the rate of degradation of the scaffoldshould ideally match the growth rate of cells and tissues Forbiodegradable polymers degradation usually takes place in 4steps water absorption reduction of mechanical properties(modulus amp strength) reduction of molar mass and weightloss due to diffusion of soluble oligomeric components [2425] Therefore we tested the scaffoldrsquos degradation by weightloss measurements Ourmeasurement of in vitro degradationmay help us understand and predict the in vivo behavior ofthese scaffolds The in vitro degradation test was performedover a span of 80 d (Figure 4(b)) and cPU showed rapiddegradation kinetics Weight loss reached 30 after the first20 d and up to 525 by day 80 which was faster than othermaterials such as PLLA and PCL [26ndash28] It is likely that thegood wettability and high water uptake greatly promote thematerialrsquos degradation

33 In Vitro Cytocompatibility and In Vivo BiocompatibilityThe cytocompatibility of the synthesized cPU was firstlymeasured via human hypopharyngeal fibroblast seeding Cellmorphology activity phenotype andprotein expressionweretested Due to its hydrophilicity cPU swelled significantlyin the cell culture medium and in the body during the invivo test As a result scanning electron microscopy (SEM)was not used for the observation of cell morphology Underlight microscopy cells were visible after primary fibroblastswere cultured on the cPU membrane for 2 d (Figure 5(a))There were many more live cells on the membrane at day14 (Figure 5(b)) Cells were stained by HampE in order tovisualize them on the membrane MTT test showed thatthe seeded cells grew and proliferated with time althoughthe mitochondrial activity (as determined by OD value) waslower than that on TCPS at every time point during the 14 dculture span (Figure 6) Mitochondrial activity on cPU at day14 only reached 36of that onTCPSThis valuewas similar tothe activity of skeletal muscle cell from human hypopharynxon poly(ester urethane) (58213 NAT 022 China) [21] Themuch higher hydrophicility of cPU resulted in the loweredcell adhesion (OD at day 1) However the increase rate of ODvalue from day 1 to day 14 is quite similar between cPU andTCPS which means the similar cell proliferation capabilitybetween these two matrices On the other hand cPU wasfound to possess rapid degradation rate from weight loss test(Figure 4(b)) It may make fibroblasts very difficult to adherefirmly on the matrix with the passing time leading to lowertest results than the reality is Despite this the cells culturedon cPUhad a fibroblast-like phenotype Vimentin is the inter-mediate filament in fibroblasts Thus it is a reliable fibroblastmarker Using anti-vimentin as the primary antibody cells


(a1) (a2) (a3)

(a)

(B1)(b2)

(B(B(B(B(B(B(BBB((B(B(BBBBBBB(BBBBB(BBBBBBBB((B((B(B(B(BB(BB(((BBBB(BB(((BBBBB(BBBBB(((BBBBBBB((((BBBBBBBBBBB(((((BBBBBBB(BBBBBBB((BBBBBBB((((BBBBBBBBBB((BBBBBB((B((BBBBBBB((B(BBBBBBBBBBBB1111111111111))))))))))))))))))) (b3)(b1)

(b)

Figure 7 Immunofluorescence of cells cultured on cPU membrane for 2 d (a) and 14 d (b) Anti-vimentin was used as the primary antibodyand appears in the cytoplasm as green fluorescenceThe nucleus is blue from DAPI stainingThe seeding density was 4 times 104 cellsmL Image(3) is an overlay of images (1) and (2) Scale bar 50120583m

TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)

Figure 8 Vimentin expression by cells seeded on cPU and TCPS for 14 d analyzed by Western blotting (a) Strips of cPU and TCPS (b)Quantitative analysis of vimentin expression Beta-actin was used as the reference to normalize the cellular protein content 119875 value is lt005

on cPU substrateswere immuno-stained (green fluorescence)to confirm the fibroblasts after they had been cultured invitro from 2 d to 14 d (Figure 7) Quantification of vimentinexpression verified that the cultured cells still had the abilityto differentiate (Figure 8) The amount of vimentin secretedby cells grown on cPUwas 35 of that of cells on TCPS at day

14 which was in agreement with the result of mitochondrialactivity analysis

In order to ascertain the in vivo biocompatibility of oursynthetic material cPU was implanted subcutaneously intothe back of SD rats After implantation for 7 21 49 and70 d SD rats were anaesthetized and the location of the cPU


(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m

Figure 9 Appearance of subcutaneously implanted cPUmembrane in the back of SD rats for 7 d (a1) 21 d (b1) 49 d (c1) and 70 d (d1) arrowindicates the location of the material (b2) HampE staining of microtome section after 21 d implantation white triangle and arrow indicatethe site filled with inflammatory cells (d2) HampE staining of microtome section after 70 d implantation Arrow indicates the biodegradedfragment Scale bar 100 120583m

membrane was exposed By visual observation the subcuta-neous samples were already enclosed in the skin hypodermisafter 7 d However a small amount of inflammation and seri-ous blood swelling were observed all around the membrane(Figure 9(a1) arrow) By day 49 the blood swelling wascomparatively reduced (Figure 9(b1) arrow) However manyinflammatory cells filled the circumference of the material(Figure 9(b2) white triangle) while some inflammatory cellshad infiltrated into the material interspace (Figure 9(b2)white arrow) The inflammation and blood swelling reducedsignificantly by day 49 (Figure 9(c1) arrow) and were com-pletely resolved by day 70 (Figure 9(d1) arrow) Angiogenesiswas observed around the sample which greatly promotes

the biocompatibility of the scaffold HampE staining revealedthe clear tissue structure (Figure 9(d2)) However during thisperiod of time the material had begun to biodegrade andsome fragments could be observed (Figure 9(d2) arrow) Weconsidered the material to have been accepted by the animalbody

4 Conclusions

A biodegradable polyurethane cPU was produced bycrosslinking HDI PEG and prepolymer PLEG which wassynthesized from the reaction between L-LA and PEGusing Fe(acac)

3as the catalyst The reaction chemistry was


followed and analyzed via FTIR spectroscopy 1H NMR andGPC The synthesized cPU possesses low 119879

119892(minus22∘C) good

hydrophilicity and relatively fast degradation It also hasthe ability to support the growth of human hypopharyngealfibroblasts Subcutaneous implantation in SD rats suggeststhat the material has good biocompatibility Howeverthe material is still weak and degrades rather quicklywhen compared with industrial polyurethane products Thesynthesis reaction and product chemistry are being improvedin our laboratory in order to meet the requirements of a goodscaffolding material for hypopharyngeal tissue engineering

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Financial support fromMajor Project Foundation of Ningbo(2012C5015) Scientific Innovation Team Project of Ningbo(2012B82019) and National Science Foundation (81171476and 81471797) of China are gratefully acknowledged Thiswork was also sponsored by the KC Wang Magna Fund ofNingbo University

References

[1] A Fortin C S Wang and E Vigneault ldquoInfluence of smokingand alcohol drinking behaviors on treatment outcomes ofpatients with squamous cell carcinomas of the head and neckrdquoInternational Journal of Radiation Oncology Biology Physics vol74 no 4 pp 1062ndash1069 2009

[2] C G Gourin and D J Terris ldquoCarcinoma of the hypopharynxrdquoSurgical Oncology Clinics of North America vol 13 no 1 pp 81ndash98 2004

[3] J Y W Chan and W I Wei ldquoCurrent management strategy ofhypopharyngeal carcinomardquo Auris Nasus Larynx vol 40 no 1pp 2ndash6 2013

[4] J Bourhis J Overgaard H Audry et al ldquoHyperfractionatedor accelerated radiotherapy in head and neck cancer a meta-analysisrdquoThe Lancet vol 368 no 9538 pp 843ndash854 2006

[5] D Perez-Smith M Wagels and D R Theile ldquoJejunal freeflap reconstruction of the pharyngolaryngectomy defect 368consecutive casesrdquo Journal of Plastic Reconstructive amp AestheticSurgery vol 66 no 1 pp 9ndash15 2013

[6] E M Genden and A S Jacobson ldquoThe role of the anterolateralthigh flap for pharyngoesophageal reconstructionrdquo Archives ofOtolaryngology Head and Neck Surgery vol 131 no 9 pp 796ndash799 2005

[7] R Liu P Gullane D Brown and J Irish ldquoPectoralis majormyocutaneous pedicled flap in head and neck reconstructionretrospective review of indications and results in 244 con-secutive cases at the Toronto General Hospitalrdquo Journal ofOtolaryngology vol 30 no 1 pp 34ndash40 2001

[8] R S Patel D P Goldstein D Brown J Irish P J Gullaneand RW Gilbert ldquoCircumferential pharyngeal reconstructionhistory critical analysis of techniques and current therapeuticrecommendationsrdquo Head and Neck vol 32 no 1 pp 109ndash1202010

[9] X Jiang J Gu L Lin and Y Zhang ldquoInvestigation on the mod-ification to polyurethane by multi-walled carbon nanotubesrdquoPigment amp Resin Technology vol 40 no 4 pp 240ndash246 2011

[10] T Yoshii A E Hafeman J M Esparza A Okawa G Gutierrezand S A Guelcher ldquoLocal injection of lovastatin in biodegrad-able polyurethane scaffolds enhances bone regeneration in acritical-sized segmental defect in rat femorardquo Journal of TissueEngineering and Regenerative Medicine vol 8 no 8 pp 589ndash595 2014

[11] A S More T Lebarbe L Maisonneuve B Gadenne C Alfosand H Cramail ldquoNovel fatty acid based di-isocyanates towardsthe synthesis of thermoplastic polyurethanesrdquo European Poly-mer Journal vol 49 no 4 pp 823ndash833 2013

[12] X Pan P Sengupta and D C Webster ldquoHigh biobased contentepoxy-anhydride thermosets from epoxidized sucrose esters offatty acidsrdquo Biomacromolecules vol 12 no 6 pp 2416ndash24282011

[13] R Slivniak and A J Domb ldquoLactic acid and ricinoleic acidbased copolyestersrdquo Macromolecules vol 38 no 13 pp 5545ndash5553 2005

[14] S A Guelcher ldquoBiodegradable polyurethanes synthesis andapplications in regenerative medicinerdquo Tissue Engineering PartB Reviews vol 14 no 1 pp 3ndash17 2008

[15] Y N Lei Y B Zhu L Chen and L X Hou ldquoPolymerization ofaliphatic cyclic ester using ironcompounds as catalystsrdquoChineseJournal of Polymer Science vol 24 pp 297ndash302 2011

[16] Y N Lei Y B Zhu C F Gong J J Lv C Kang and L X HouldquoSynthesis characterization and cytocompatibility of a noveldegradable polymer using iron compound as catalystrdquo Journalof Materials Science Materials in Medicine vol 2 pp 273ndash2822014

[17] A M Haaparanta E Jarvinen I F Cengiz et al ldquoPreparationand characterization of collagenPLA chitosanPLA and colla-genchitosanPLA hybrid scaffolds for cartilage tissue engineer-ingrdquo Journal of Materials Science Materials in Medicine vol 25no 4 pp 1129ndash1136 2014

[18] X Zhong C Ji A K L Chan S G Kazarian A Ruys and FDehghani ldquoFabrication of chitosanpoly(120576-caprolactone) com-posite hydrogels for tissue engineering applicationsrdquo Journal ofMaterials Science Materials in Medicine vol 22 no 2 pp 279ndash288 2011

[19] G-M Kim KH T Le SMGiannitelli Y J Lee A Rainer andM Trombetta ldquoElectrospinning of PCLPVP blends for tissueengineering scaffoldsrdquo Journal of Materials Science Materials inMedicine vol 24 no 6 pp 1425ndash1442 2013

[20] P Jithendra A M Rajam T Kalaivani A B Mandal and CRose ldquoPreparation and characterization of aloe vera blendedCollagen-Chitosan composite scaffold for tissue engineeringapplicationsrdquo ACS Applied Materials amp Interfaces vol 5 no 15pp 7291ndash7298 2013

[21] Z Shen S Guo D Ye et al ldquoSkeletal muscle regenerationon protein-grafted and microchannel-patterned scaffold forhypopharyngeal tissue engineeringrdquo BioMed Research Interna-tional vol 2013 Article ID 146953 8 pages 2013

[22] A K Gaharwar C Rivera C-J Wu B K Chan and GSchmidt ldquoPhotocrosslinked nanocomposite hydrogels fromPEG and silica nanospheres structural mechanical and celladhesion characteristicsrdquo Materials Science and Engineering CMaterials for Biological Applications vol 33 no 3 pp 1800ndash18072013

[23] AW Smith J DHoyne P K Nguyen et al ldquoDirect reprogram-ming of mouse fibroblasts to cardiomyocyte-like cells using


Yamanaka factors on engineered poly(ethylene glycol) (PEG)hydrogelsrdquo Biomaterials vol 34 no 28 pp 6559ndash6571 2013

[24] M Hakkarainen A C Albertsson and S Karlsson ldquoWeightlosses and molecular weight changes correlated with the evolu-tion of hydroxyacids in simulated in vivo degradation of homo-and copolymers of PLA and PGArdquo Polymer Degradation andStability vol 52 no 3 pp 283ndash291 1996

[25] A Gopferich ldquoMechanisms of polymer degradation and ero-sionrdquo Biomaterials vol 17 no 2 pp 103ndash114 1996

[26] A Hoglund M Hakkarainen and A-C Albertsson ldquoMigra-tion and hydrolysis of hydrophobic polylactide plasticizerrdquoBiomacromolecules vol 11 no 1 pp 277ndash283 2010

[27] J E Bergsma W C de Bruijn F R Rozema R R M Bos andG Boering ldquoLate degradation tissue response to poly(L-lactide)bone plates and screwsrdquo Biomaterials vol 16 no 1 pp 25ndash311995

[28] P Shokrollahi M Mehmanchi M Atai H Omidian and FShokrolahi ldquoEffect of interface on mechanical properties andbiodegradation of PCLHAp supramolecular nano-compositesrdquoJournal of Materials Science Materials in Medicine vol 25 no1 pp 23ndash35 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 The component feeding ratios and GPC measurements of prepolymer PLEG

Copolymer PEGL-LA in feeding (mol) 119872119899(Da) 119872

119908(Da) 119872

119908119872119899

PLEG 1 12 1 4365 5926 13576PLEG 2 1 1 4702 5977 12709PLEG 3 1 2 5422 6311 11638PLEG 4 1 5 6046 7312 12090

with in the production of thermoplastic and biodegradablepolyurethanes [11ndash14] In our previous work we studied thecatalysis of aliphatic ester polymerization by low toxicityiron compounds instead of the stannum compounds usedcommonly in research orand industry [15] Using thisapproach we synthesized a biocompatible and biodegradablecopolymer with 119879

119892of 56∘C from monomers of L-lactide

poly(ethylene glycol) (PEG) and NIPAAm [16] Howeverthe previously published polymer was too rigid to be usedin reconstruction of soft tissues like the hypopharynx Inthis study we have produced a material with lower 119879

119892

better strength and superior biodegradability using reactionsbetween L-lactide PEG and hexamethylene diisocyanate(HDI) The frequency and chemistry of the reaction werecharacterized by Fourier transform infrared spectroscopy(FTIR) Hydrogen-1 nuclear magnetic resonance (1H NMR)spectra and Gel Permeation Chromatography (GPC) Thenovel materialrsquos mechanical property hydrophilicity degrad-ability cytocompatibility and in vivo biocompatibility wereevaluated We believe that the material presented in thispaper will be a good substitute for hypopharyngeal tissueengineering

2 Materials and Methods

21 Materials PEG (Mn 2000) dichloromethane (CH2Cl2)

ethanol and ethyl acetate were purchased from SinopharmChemical Reagent Co Ltd L-lactide (L-LA) was supplied byJinan Daigang Biological Material Company It was recrystal-lized three times using ethyl acetate as the solvent prior toreaction Iron (III) acetylacetonate (Fe(acac)

3) and HDI were

purchased from Aladdin Reagent Co Ltd ChinaTrypsin (1 250 GNM) was purchased from Beijing

Genosys Scientific Co China Mouse anti-vimentin andFITC-conjugated goat anti-mouse IgG were from WuhanBoster Bio-Engineering Co Ltd IRDye 680RD goat anti-mouse IgG (H+L) was purchased from LI-COR BiosciencesUSA All cell culture reagents were purchased from HyCloneunless otherwise specified All chemical reagents used forWestern blotting were purchased from Beyotime Institute ofBiotech Jiangsu China Phosphate buffer saline (PBS pH74)used in cell culture was sterilized prior to use Sprague-Dawley (SD) male rats were obtained from the ExperimentalAnimal Center of Ningbo University

22 Synthesis of PLA-PEG-PLA Prepolymer (PLEG) Theprepolymer PLEGwas synthesized using L-LA and PEG (Mn2000) as monomers and Fe(acac)

3as the catalyst as per our

previous protocol [12] Briefly predetermined amounts of

PEG and L-LA were added into a polymerization tube withFe(acac)

3(05 weight ratio to total reactants resolved in

CH2Cl2) as the catalyst After degassing for 2 h at 60∘C the

tube was sealed under vacuum and placed entirely into an oilbath and the bath temperature was maintained at 130∘C for20 h with stirring The polymerization tube was then takenout of the oil bath and cooled to room temperature Tenmilliliters CH

2Cl2was added to dissolve the product After

precipitation in ethanol at 0∘C the three-block PLA-PEG-PLA prepolymer (PLEG) with different ratios of monomerswas obtained (Table 1) The PLEG was subsequently dried ina vacuum oven and kept in the dryer for the next reaction

23 Preparation of Crosslinked Polyurethane PrepolymerPLEG PEG and HDI (molar ratio 165 10 29) were mixedand heated to 80∘C for 8 h under nitrogen protection toallow crosslinking to occur PLEG and PEG were sepa-rately dehydrated via degassing for 1 h at 110∘C prior to thecrosslinking reaction A shallow yellow sticky solution wasformed at the end of the reaction It was dissolved in CH

2Cl2

to produce a 50 (v v) solution which was cast onto apolydimethylsiloxane (PDMS) film and dried in an oven at60∘C overnight to yield a flat approximately 10mm thickcPU membrane PDMS was used as the substrate as thesynthesized cPU membrane could easily be peeled off

24 Characterization of Prepolymer and Crosslinked Polymer

241 PLEGPrepolymer FTIR spectrawere recorded using anFTIR instrument (Digilab FTS 3100 USA) with 4 cmminus1 reso-lution and a measuring period of 500ndash4000 cmminus1 The reac-tion chemistry of prepolymer PLEG was confirmed via1H NMR measurement PLEG sample was dissolved inCDCl

3and tested on a NMR spectrometer (Bruker Avance

400MHz Switzerland) The molecular weight (Mn and Mw)and molecular weight distribution (MwMn) were deter-mined by GPC (Polymer Laboratories PL-GPC 50 plusEngland) using polystyrene as the standard The analysis wasperformed at 40∘C using tetrahydrofuran (THF) as the eluentat a flow rate of 10mLmin

242 Structure and Characteristics of cPU The thermalproperties of cPU were tested by differential scanningcalorimetry (DSC PyrisDiamondUSA) under nitrogenThefirst heating was from 25 to 100∘C at a rate of 20∘Cmin with1min station to clear the thermal history then the samplewas cooled down to minus50∘C at a rate of 10∘Cmin The secondheating started from minus50∘C and increased to 100∘C at a rate of20∘Cmin The glass transition temperature (119879

119892) and melting















1198820

times 100 (1)





















3at




3)CHCO-







H H

O

O

O

C

C

OOH

PEG

(b)

(a)

L-LA

OCH

O O

CH OH

PLA-PEG-PLA (PLEG)

OCCOCH2CH2 OCH2CH2n

x

n

y

+Fe(acac)3

135∘CCH3 CH3


CH2CH2

simsimsimsim

simsimsimsim

simsimsimsim

simsimsimsim



CO-N

H-(CH

2 )6 -HN

-CO-

-




119879119892(∘C) 119879



050

100150200250300350400450500

Tran

smitt

ance

2270

1756

3507 33202866 1750

16271262

1094

(a)

(b)

(c)

(d)

(e)

4000 3500 3000 2500 2000 1500 1000 500


minus100

minus50



H OCH CO

OC CH

x

n

y

OHO

(a)

(b)(c)

1 2 3 4 5 6(ppm)

(b) 364

PLEGCH3

CH3

OCH2CH2

(c) 517

(a) 157Δ316

Δ100

Δ710




119892)





0 10 20 30 40 50 60 70 80

0

1

2

3

4

5

Stre

ss (M

Pa)

Strain ()

(a)

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Stre

ss (M

Pa)

Strain ()minus20

(b)


40

0 50 100 150 200

45

50

55

60

65

70

75

Time (s)

Dyn

amic

cont

act a

ngle

(deg

)

(a)

40

30

20

10

0 20 40 60 80

50

60W

eigh

t los

s (

)

Time (d)

(b)


(a) (b)



00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145







(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























1198820

times 100 (1)





















3at




3)CHCO-







H H

O

O

O

C

C

OOH

PEG

(b)

(a)

L-LA

OCH

O O

CH OH

PLA-PEG-PLA (PLEG)

OCCOCH2CH2 OCH2CH2n

x

n

y

+Fe(acac)3

135∘CCH3 CH3


CH2CH2

simsimsimsim

simsimsimsim

simsimsimsim

simsimsimsim



CO-N

H-(CH

2 )6 -HN

-CO-

-




119879119892(∘C) 119879



050

100150200250300350400450500

Tran

smitt

ance

2270

1756

3507 33202866 1750

16271262

1094

(a)

(b)

(c)

(d)

(e)

4000 3500 3000 2500 2000 1500 1000 500


minus100

minus50



H OCH CO

OC CH

x

n

y

OHO

(a)

(b)(c)

1 2 3 4 5 6(ppm)

(b) 364

PLEGCH3

CH3

OCH2CH2

(c) 517

(a) 157Δ316

Δ100

Δ710




119892)





0 10 20 30 40 50 60 70 80

0

1

2

3

4

5

Stre

ss (M

Pa)

Strain ()

(a)

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Stre

ss (M

Pa)

Strain ()minus20

(b)


40

0 50 100 150 200

45

50

55

60

65

70

75

Time (s)

Dyn

amic

cont

act a

ngle

(deg

)

(a)

40

30

20

10

0 20 40 60 80

50

60W

eigh

t los

s (

)

Time (d)

(b)


(a) (b)



00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145







(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3at




3)CHCO-







H H

O

O

O

C

C

OOH

PEG

(b)

(a)

L-LA

OCH

O O

CH OH

PLA-PEG-PLA (PLEG)

OCCOCH2CH2 OCH2CH2n

x

n

y

+Fe(acac)3

135∘CCH3 CH3


CH2CH2

simsimsimsim

simsimsimsim

simsimsimsim

simsimsimsim



CO-N

H-(CH

2 )6 -HN

-CO-

-




119879119892(∘C) 119879



050

100150200250300350400450500

Tran

smitt

ance

2270

1756

3507 33202866 1750

16271262

1094

(a)

(b)

(c)

(d)

(e)

4000 3500 3000 2500 2000 1500 1000 500


minus100

minus50



H OCH CO

OC CH

x

n

y

OHO

(a)

(b)(c)

1 2 3 4 5 6(ppm)

(b) 364

PLEGCH3

CH3

OCH2CH2

(c) 517

(a) 157Δ316

Δ100

Δ710




119892)





0 10 20 30 40 50 60 70 80

0

1

2

3

4

5

Stre

ss (M

Pa)

Strain ()

(a)

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Stre

ss (M

Pa)

Strain ()minus20

(b)


40

0 50 100 150 200

45

50

55

60

65

70

75

Time (s)

Dyn

amic

cont

act a

ngle

(deg

)

(a)

40

30

20

10

0 20 40 60 80

50

60W

eigh

t los

s (

)

Time (d)

(b)


(a) (b)



00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145







(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H H

O

O

O

C

C

OOH

PEG

(b)

(a)

L-LA

OCH

O O

CH OH

PLA-PEG-PLA (PLEG)

OCCOCH2CH2 OCH2CH2n

x

n

y

+Fe(acac)3

135∘CCH3 CH3


CH2CH2

simsimsimsim

simsimsimsim

simsimsimsim

simsimsimsim



CO-N

H-(CH

2 )6 -HN

-CO-

-




119879119892(∘C) 119879



050

100150200250300350400450500

Tran

smitt

ance

2270

1756

3507 33202866 1750

16271262

1094

(a)

(b)

(c)

(d)

(e)

4000 3500 3000 2500 2000 1500 1000 500


minus100

minus50



H OCH CO

OC CH

x

n

y

OHO

(a)

(b)(c)

1 2 3 4 5 6(ppm)

(b) 364

PLEGCH3

CH3

OCH2CH2

(c) 517

(a) 157Δ316

Δ100

Δ710




119892)





0 10 20 30 40 50 60 70 80

0

1

2

3

4

5

Stre

ss (M

Pa)

Strain ()

(a)

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Stre

ss (M

Pa)

Strain ()minus20

(b)


40

0 50 100 150 200

45

50

55

60

65

70

75

Time (s)

Dyn

amic

cont

act a

ngle

(deg

)

(a)

40

30

20

10

0 20 40 60 80

50

60W

eigh

t los

s (

)

Time (d)

(b)


(a) (b)



00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145







(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 20 30 40 50 60 70 80

0

1

2

3

4

5

Stre

ss (M

Pa)

Strain ()

(a)

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Stre

ss (M

Pa)

Strain ()minus20

(b)


40

0 50 100 150 200

45

50

55

60

65

70

75

Time (s)

Dyn

amic

cont

act a

ngle

(deg

)

(a)

40

30

20

10

0 20 40 60 80

50

60W

eigh

t los

s (

)

Time (d)

(b)


(a) (b)



00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145







(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

02

04

06

08

10

12

14

OD

Time (d)

TCPScPU

1 10 145







(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a1) (a2) (a3)

(a)

(B1)(b2)


(b)


TCPScPU

(a)

0

1

2

3

4

5

P lt 005

cPU TCPS

(b)






(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a1)

(c1) (d1)

(b1)

(b2) (d2)

100120583m100120583m




4 Conclusions









Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Synthesis and Characterization of Biodegradable...

Documents

Transcript of Research Article Synthesis and Characterization of Biodegradable...