research article synthesis of functional polyester based on polylactic acid...

Research ArticleSynthesis of Functional Polyester Based on Polylactic Acid andIts Effect on PC12 Cells after Coupling with Small Peptides

Na Qiang1 Shuo Tang2 Xiang Liao2 Hao Liang1 Fang Xie1 and Ji-xiang Zhu3

1Department of Chemical Engineering Huizhou University Huizhou 516007 China2Department of Pain Medicine Nanshan Hospital Shenzhen 51700 China3Department of Biomedical Engineering Guangzhou Medical University Guangzhou 510182 China

Correspondence should be addressed to Shuo Tang tangshuo1205163com

Received 7 January 2016 Revised 15 May 2016 Accepted 13 June 2016

Academic Editor Cornelia Vasile

Copyright copy 2016 Na Qiang et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Polyesters containing functional groups are a suitable candidate matrix for cell culture in tissue engineering Three types ofsemicrystalline copolymer poly(l-lactide-co-120573-malic acid) [P(LA-co-BMD)] with pendent carboxyl groups were synthesized inthis study The functional monomer 3(S)-[(benzyloxycarbonyl)methyl]-14-dioxane-25-dione (BMD) was synthesized using l-aspartic acidThe copolymer P(LA-co-BMD)was then synthesized through ring-opening copolymerization of l-LA andBMDwithdodecanol as initiator and stannous octoate as catalyst Copolymer structure was characterized by 1H nuclear magnetic resonance(1H NMR) gel permeation chromatography (GPC) and differential scanning calorimetry (DSC) analyses Results of 1H NMRand GPC analyses showed that the copolymers were synthesized successfully DSC curves showed that the crystal melting peakand enthalpy decreased with increased BMD The crystallinity of the copolymer was destroyed by the presence of the functionalmonomer After deprotection carboxyl groups were coupled with the isoleucine-lysine-valine-alanine-valine peptide through N-hydroxysuccinimidedicyclohexylcarbodiimide method The small peptide was beneficial to the axon growth of PC12 cells

1 Introduction

Multiple synthetic biopolymers including polyglycolic acidpolylactic acid poly(lactic-co-glycolic acid) (PLGA) andpoly(caprolactone) are used as biomedical polymeric mate-rials in tissue engineering and drug delivery because of theirgood biocompatibility and biodegradability [1ndash5] Howeverthese biodegradable polymers are hydrophobic Thereforethe wettability of these materials must be enhanced throughfunctionalization Synthetic polyesters are also unsuitablefor polymer-protein interaction because of their insufficientchemical functionalities Functional polymers have beensynthesized using hydrophilic groups to satisfy the designcriteria for advanced applications These studies promotedthe coupling reaction to covalently immobilize biologicallyactive peptides onto the scaffolds Peptide-modified surfaceshave gained increased attention because surface-immobilizedbiologically active cues can lead to cell adhesion migrationor differentiation Laminin-derived peptides such as isoleu-cine-lysine-valine-alanine-valine (IKVAV) can promote cell

adhesion and induce neurite outgrowth for neural progenitorcells [6ndash11]

Copolymerization between functional monomer andpolyestermatrix introduces functional groups throughwhicha small peptide can be bonded onto the polymer Thismethod can promote cell growth and differentiation [12ndash16] Polymaleic acid (PMA) is a water-soluble polyester com-pound whose side chain contains the carboxyl group PMAis metabolized in vivo and generates malic acid which isan intermediate of the Krebs tricarboxylic acid cycle [17]Malic acid polymers with bacterial polyesters constitute anattractive material for tissue engineering These copolymerswith lateral groups can be used to couple with small peptides[18ndash21]

In this study the cyclic monomer 3(S)-[(benzyloxycar-bonyl)methyl]-14-dioxane-25-dione (BMD) was used forring-opening polymerization with l-lactide (l-LA) BMDwas synthesized through a four-step reaction with l-asparticacid as raw material The copolymerization behavior of thefunctional monomer BMD and l-LA was investigated and

Hindawi Publishing CorporationInternational Journal of Polymer ScienceVolume 2016 Article ID 9829757 6 pageshttpdxdoiorg10115520169829757

2 International Journal of Polymer Science

O

O

O

OO

O +O

O

O

O

O O O O O

O

O

O

m

O

O O

Hn

CH3

CH3

CH3(CH2)10CH2OHSn(Oct)2

H3C(H2C)10H2C

Scheme 1 Sn(Oct)2-catalyzed ring-opening polymerization of BMD with l-LA

characterized by 1H nuclear magnetic resonance (1H NMR)gel permeation chromatography (GPC) and differentialscanning calorimetry (DSC) analyses Moreover the shortIKVAV peptide was chemically bonded using functionalmonomers on the copolymer film containing carboxyl groupin the side chain The effect of short IKVAV peptide on PC12cell behavior was also determined

2 Materials and Methods

21 Materials Benzyl alcohol (C6H5CH2OH) ethyl alco-

hol (CH3CH2OH) ether (CH

3CH2OCH3CH2) ethyl ace-

tate (CH3COOC

2H5) dodecanol (C

12H25OH) pyridine

(C5H5N) sodium bicarbonate (NaHCO

3) and sodium

nitrite (NaNO2) were purchased from Guangzhou Chemical

Reagent Factory (China) l-Aspartic acid was obtained fromShanghai GiAo Biochemical Co Ltd (China) Bromoace-tyl chloride N-hydroxysuccinimide (NHS) and NN1015840-dicy-clohexylcarbodiimide (DCC) were purchased from AcrosOrganics Triethylamine fromGuangzhou Chemical ReagentFactory was distilled from CaH

2 NN-Dimethylformamide

(DMF) was distilled from 4 A molecular sieve l-LA wasobtained from Huizhou Foryou Medical Device Co Ltd(Guangdong China) and recrystallized three times in ethylacetate Stannous octoate [Sn(Oct)

2] was obtained from Alfa

Aesar Trifluoroacetic acid was purchased from AldrichHydrogen bromide (HBr) in acetic acid (33) was purchasedfrom Acros Organics IKVAV peptides were synthesized byGL Biochem Co Ltd

22 Characterization The mole ratio of l-LA to BMD wasanalyzed by 1H NMR spectroscopy with CDCl

3as solvent

and tetramethylsilane as internal standard Fourier transforminfrared (FTIR) spectra were obtained by NicoletNexus 670Number-average (119872

119899) molecular weights were determined

using GPC (Waters 410 Milford MA) equipped with organicGPC columns Chloroform was used as mobile phase at aflow rate of 10mLmin at 35∘C Thermal properties wereexamined by DSC and the samples were heated from 0∘Cto 200∘C at a heating rate of 10∘Cmin under nitrogenatmosphere Cell morphology was studied using scanningelectron microscopy (SEM JSM-6380LA Analytical JEOLLtd Tokyo Japan) equippedwith vacuummode and at 15 kVimaging

23 Copolymer Synthesis

231 Copolymerization of l-LA with BMD BMD was syn-thesized using l-aspartic acid through a four-step reactionDifferent molar ratios of l-LA and BMD (982 955 and928) were added to a dry Schlenk tube under dry Ar atmo-sphere Dodecanol was added as initiator ([119872][119868] = 7001)with Sn(Oct)

2as catalyst ([119872][119868] = 10001) The tube was

evacuated for 2 h at ambient temperature sealed immersedin an oil bath and heated at 110∘C for 48 h The three typesof copolymers were dissolved in chloroform and precipitatedwith methanol (Scheme 1)

232 Deprotection of Copolymers to Obtain Carboxyl-Sub-stituted Polymers Copolymers with protecting groups (2 g)were dissolved in trifluoroacetic acid (20mL) Briefly 33HBrCH

3COOH solution (8mL) was added dropwise under

Ar atmosphere after the complete dissolution of the copoly-mer The product was poured into anhydrous ether (200mL)after 5 h at ambient temperature The precipitates were dis-solved in chloroform and purified by ethanol

24 Modification of Copolymer Films by Short Peptides Co-polymers after deprotection (2 g) were dissolved in chlo-roform (30mL) NHS (3mmol 06 g) and DCC (3mmol034 g) were added dropwise The compounds were reactedfor 2 h in an ice bath and for 24 h at room temperature Theproduct was dissolved in ethanol and precipitated withchloroform

The polymer films were prepared by solution pouringand solvent evaporationThe samples were then immersed in10mgmL short-peptide solution after drying The film wasthen soaked for 12 h with mild agitation and washed threetimes (once per hour) with phosphate-buffered saline (PBS)solution to remove unreacted short peptides

25 Cell Culture and Morphology Observation The polymerfilm functionalized with peptide on 15mm cover slip wasplaced in a 24-well plate and pressed with ring to ensure com-plete contact of the scaffolds to the wellsThe specimens weresterilized under UV light washed three times with PBS andimmersed in Dulbeccorsquos modified Eaglersquos medium (DMEM)overnight before cell seeding Rat pheochromocytoma-derived cell line (PC12) was seeded on the film at a densityof 10 times 104 cellswell and cultured in DMEM containing

International Journal of Polymer Science 3

CH3

120575 (ppm)8 7 6 5 4 3 2 1

10 09 08

(h + c) (g)

(f) (e) (d)

(b)

O O OO O

O

O

O

m

O

O O

Hn

(a)

(b)

(c)

(d)

(e) (f) (h)

(g)

(h)

(g)

(a)

CH3

H3C(H2C)10H2C

Figure 1 1H NMR spectrum of P(LA92-co-BMD

8) random copolymers (CDCl

3)

Table 1 Polymerization of l-LA with BMD initiated by dodecanol and catalyzed with stannous octoatea

l-LAtheoBMD l-LAHNMRbBMD 119872

119899

c (gmol) 119872119899

c (gmol) 119872119908119872119899

c 119879119892

d (∘C) 119879119898

d (∘C) Δ119867119898

d (Jg)1000 1000 mdash mdash mdash 608 1772 936982 98020 10 times 105 16 times 105 103 559 1624 556955 95446 10 times 105 11 times 105 112 537 1542 548928 93664 10 times 105 10 times 105 119 534 1474 94aPolymerization of BMD with l-LA at 100∘C for 48 hbObtained from 1H NMR analysiscNumber-average molar mass (119872119899) and polydispersity index (119872119908119872119899) obtained from gel permeation chromatography in THF using polystyrene standardsdThe thermal properties were determined using DSC at 10∘Cmin heating rate under N2

10 FBS heat-inactivated fetal bovine serum and 1 peni-cillinstreptomycin at 37∘C in a humidified atmosphere of95 air and 5CO

2The culturemediumwas replaced every

3 days Tissue culture polystyrene was used as controlThe cell-cultured films were processed for SEM studies 5

days after cell proliferation The scaffolds were washed twicewith PBS and fixed in 3 glutaraldehyde for 3 hThe scaffoldswere then washed with deionized water and ethanol (5070 90 and 100 concentrations) twice for 15min eachFinally the films were coated with gold and observed by SEManalyses

3 Results and Discussions

31 Characterization of the Copolymers The results of the 1HNMR spectrum andGPC test showed the successful synthesis

of the copolymer poly(l-lactide-co-120573-malic acid) [P(LA-co-BMD)] The 1HNMR spectrum of the copolymer P(LA-co-BMD) is shown in Figure 1 The resonance absorptionassigned to the PlLA segment was observed at 12057516 ppm (g3H CH

3) and 12057551 ppm (h 1H OCH) after the ring-opening

polymerization of l-LA The peaks at 12057546 12057551 12057555 and12057573 ppm could be attributed to the resonance absorptionof the PBMD segment The proportion of the functionalmonomers in the copolymer was calculated using the ratioof relative integral intensity of resonance peak in the 1HNMR spectrum In particular the integral intensity ratio ofthe characteristic peak (g) was calculated after ring-openingpolymerization of l-LA to the peak (b) for BMD unit thatis (119868g6)(119868b5) The molar ratios of PlLA to PBMD werein agreement with the feed ratios (Table 1) However theactual ratios of l-LABMD in the copolymer to the feed ratiosincreased with the increase in feed ratio of BMD


3500 3000 2500 2000 1500 1000 500

(a)

(b)

Wavenumbers (cmminus1)

1739 cmminus11708 cmminus1

1728 cmminus1

Figure 2 Infrared spectra of (a) BMD and (b) after ring-openingpolymerization

10 11 12 13 14 15 16 17

(c)(b)

Elution time (min)

(a)

Figure 3 Gel permeation chromatography traces of the obtainedP(LA-co-BMD) (a) P(LA

98-co-BMD

2) (b) P(LA

95-co-BMD

5) and

(c) P(LA92-co-BMD

8)

Figure 2 shows the FTIR spectra over the range 4000ndash500 cmminus1 for the BMD and ring-opening of BMD The spec-tra (Figure 2(a)) showed the carboxyl and hydroxyl band forBMD at 2968 and 3504 cmminus1 respectively The characteristicbands at 1739 and 1708 cmminus1 were the carbonyl bands ofBMD After ring-opening polymerization (Figure 2(b)) thecarbonyl band of PBMD on the main chain appeared at1728 cmminus1 The carbonyl bands of BMD (1739 and 1708 cmminus1)decreased The results indicated the ring-opening polymer-ization of BMD

The 119872119899values of polymers P(LA

98-co-BMD

2) P(LA

95-

co-BMD5) and P(LA

92-co-BMD

8) were 16 times 105 11 times 105

and 10 times 105 respectively The polymer distributions ofmolecular weights were 103 112 and 119119872

119899decreased and

the distribution of the molecular weights widened (Table 1)This result could probably be due to the interaction betweenbenzyl groups and stannous octoate [22] The GPC curves ofthe copolymer with different ratios are shown in Figure 3

0 50 100 150

(c)

(b)

Endo (a)

T (∘C)

Figure 4 Differential scanning calorimetry thermograms of theobtained P(LA-co-BMD) (a) P(LA

98-co-BMD

2) (b) P(LA

95-co-

BMD5) and (c) P(LA

92-co-BMD

8)

32Thermal Properties of the Copolymers The thermal prop-erties of the copolymers were investigated by DSC mea-surement (Figure 4) The endothermic peaks between 147∘Cand 162∘C could be attributed to the melting transitionof the crystalline phase in the second-heating runs Theendothermic peaks between 53∘C and 55∘C could be ascribedto the glass transition temperature of the polymer 119879

119898 119879119892

and ΔH of the polymer were lower than those of pure PlLAand decreased with the increase in molar ratio of BMDThe reason could be that the crystallization was significantlydisturbed by the functional BMD

33 Adhesion and Morphology of Cells on Films Modified byShort Peptides The SEM images of the PC12 cells after 5 daysof seeding are shown in Figure 5 PC12 cells showed betteradhesion and proliferation because of the film modificationby small peptide PC12 cells showed spheroid body at PPlLA film At 5 days after cell seeding PC12 cells overthe film modified with small peptide (IKVAV) spread outHowever the effect of different polymer materials on cellbehavior was different The functional group on the threetypes of copolymers was different and thus the coupling withpeptide was differentThe axon length of the cells on P(LA

98-

co-BMD2) film was less than that of the cells on P(LA

95-

co-BMD5) and P(LA

92-co-BMD

8) However no significant

difference between the axon lengths of the cells on P(LA95-co-

BMD5) and P(LA

92-co-BMD

8) was observed Therefore the

effect of short-peptide density on cell behavior was significantin a certain density range The results are consistent with theresults found in literature [23]

4 Conclusions

In this study a series of PlLA-based functional copolymerscontaining different density of functional monomer (BMD)were synthesized and were characterized The structure ofBMD with pendent carboxyl groups was used for coupling


dP(LA98-co-BMD2) P(LA95-co-BMD5) P(LA92-co-BMD8)PLLA

Figure 5 Scanning electron micrographs of PC12 cells seeded on the copolymeric films 5 days after isoleucine-lysine-valine-alanine-valinelinking

with small peptide IKVAV The effects of the density ofpeptide IKVAVonPC12 cells axon growthwere exploredTheaxon length of PC12 cells on the polymeric surface of P(LA

95-

co-BMD5) and P(LA

92-co-BMD

8) was significantly longer

than that of P(LA98-co-BMD

2) These results demonstrated

higher density of peptide IKVAV can promote axon growthof PC12 cells and the biomimetic polymers have potentialapplication as biomaterials in tissue engineering

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the National Youth ScienceFund (81401787) the National Natural Science Foundation ofGuangdong China (2015A030310353 2015A030310345)and the Science Program of Huizhou University(20141111103042712)

References

[1] Z Pan and J D Ding ldquoPoly(lactide-co-glycolide) porous scaf-folds for tissue engineering and regenerative medicinerdquo Inter-face Focus vol 2 no 3 pp 366ndash377 2012

[2] N Pirooznia S Hasannia A S Lotfi and M Ghanei ldquoEncap-sulation of alpha-1 antitrypsin in PLGA nanoparticles in vitrocharacterization as an effective aerosol formulation in pul-monary diseasesrdquo Journal of Nanobiotechnology vol 10 article20 pp 1ndash15 2012

[3] Z Sheikh S Najeeb Z Khurshid V Verma H Rashid and MGlogauer ldquoBiodegradable materials for bone repair and tissueengineering applicationsrdquo Materials vol 8 no 9 pp 5744ndash5794 2015

[4] R Smeets F Gerhards J Stein et al ldquoA novel hemostatic deliv-ery device for thrombin biodegradable poly(DL-lactide-co-glycolide) 5050 microspheresrdquo Journal of Biomedical MaterialsResearch Part A vol 96 no 1 pp 177ndash185 2011

[5] I Armentano M Dottori E Fortunati S Mattioli and J MKenny ldquoBiodegradable polymer matrix nanocomposites fortissue engineering a reviewrdquo Polymer Degradation and Stabilityvol 95 no 11 pp 2126ndash2146 2010

[6] R J Pounder and A P Dove ldquoTowards poly(ester) nanoparti-cles recent advances in the synthesis of functional poly(ester)s

by ring-opening polymerizationrdquo Polymer Chemistry vol 1 no3 pp 260ndash271 2010

[7] Q Yin L C Yin H Wang and J J Cheng ldquoSynthesis and bio-medical applications of functional poly(120572-hydroxy acids) viaring-opening polymerization of O-carboxyanhydridesrdquo Ac-counts of Chemical Research vol 48 no 7 pp 1777ndash1787 2015

[8] R J Pounder and A P Dove ldquoSynthesis and organocatalyticring-opening polymerization of cyclic esters derived from l-malic acidrdquo Biomacromolecules vol 11 no 8 pp 1930ndash19392010

[9] O T Boullay N Saffon J-P Diehl B Martin-Vaca and DBourissou ldquoOrgano-catalyzed ring opening polymerization of a14-dioxane-25-dione deriving from glutamic acidrdquo Biomacro-molecules vol 11 no 8 pp 1921ndash1929 2010

[10] Y Yu J Zou and C Cheng ldquoSynthesis and biomedical applica-tions of functional poly(120572-hydroxyl acid)srdquo Polymer Chemistryvol 5 no 20 pp 5854ndash5872 2014

[11] W F Dai Y Y He H Y Huang and M D Lang ldquoSynthesisand characterization of poly(120576-caprolactone) bearing pendantfunctional grouprdquo Acta Polymerica Sinica vol 4 pp 358ndash3622009

[12] T Kajiyama T Taguchi H Kobayashi K Kataoka and JTanaka ldquoSynthesis of high molecular weight poly(120572120573-malicacid) for biomedical use by direct polycondensationrdquo PolymerDegradation and Stability vol 81 no 3 pp 525ndash530 2003

[13] G Barouti C G Jaffredo and S M Guillaume ldquoLinear andthree-arm star hydroxytelechelic poly(benzyl 120573-malolacto-nate)s a straightforward one-step synthesis through ring-opening polymerizationrdquo Polymer Chemistry vol 6 no 32 pp5851ndash5859 2015

[14] P Manitchotpisit C D Skory S W Peterson N P J PriceK E Vermillion and T D Leathers ldquoPoly(120573-L-malic acid)production by diverse phylogenetic clades of Aureobasidiumpullulansrdquo Journal of Industrial Microbiology amp Biotechnologyvol 39 no 1 pp 125ndash132 2012

[15] D Yao G J Li T Kuila et al ldquoLipase-catalyzed synthesis andcharacterization of biodegradable polyester containing l -malicacid unit in solvent systemrdquo Journal of Applied Polymer Sciencevol 120 no 2 pp 1114ndash1120 2011

[16] N M Kumar S K Gupta D Jagadeesh K Kanny and F BuxldquoDevelopment of poly(aspartic acid-co-malic acid) compositesfor calcium carbonate and sulphate scale inhibitionrdquo Environ-mental Technology vol 36 no 10 pp 1281ndash1290 2015

[17] Y A Zhang C H Ni G Shi J Wang M Zhang and W LildquoThe polyion complex nano-prodrug of doxorubicin (DOX)with poly(lactic acid-co-malic acid)-block-polyethylene glycolpreparation and drug controlled releaserdquo Medicinal ChemistryResearch vol 24 no 3 pp 1189ndash1195 2015


[18] Y Zeng Y Zhang and M Lang ldquoSynthesis and characteriza-tion of poly(120576-caprolactone-co-120575- valerolactone) with pendantcarboxylic functional groupsrdquoChinese Journal of Chemistry vol29 no 2 pp 343ndash350 2011

[19] X W Guan X Y Peng J Cao B He and Z W Gu ldquoSyn-thesis and cytocompatibility of biodegradable poly (L-lactide-r-5-hydroxyl trimethylene carbonate) copolymerrdquo Journal ofMacromolecular Science Part A-Pure andAppliedChemistry vol52 no 3 pp 218ndash225 2015

[20] P Loyer and S Cammas-Marion ldquoNatural and synthetic poly(malic acid)-based derivates a family of versatile biopolymersfor the design of drug nanocarriersrdquo Polymer Chemistry vol 5pp 5854ndash5872 2014


[22] W W Gerhardt D E Noga K I Hardcastle A J GarcıaD M Collard and M Weck ldquoFunctional lactide monomersmethodology and polymerizationrdquo Biomacromolecules vol 7no 6 pp 1735ndash1742 2006

[23] M Suzuki S Itoh I Yamaguchi et al ldquoTendon chitosan tubescovalently coupled with synthesized laminin peptides facilitatenerve regeneration in vivordquo Journal of Neuroscience Researchvol 72 no 5 pp 646ndash659 2003

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


O

O

O

OO

O +O

O

O

O

O O O O O

O

O

O

m

O

O O

Hn

CH3

CH3

CH3(CH2)10CH2OHSn(Oct)2

H3C(H2C)10H2C

Scheme 1 Sn(Oct)2-catalyzed ring-opening polymerization of BMD with l-LA

characterized by 1H nuclear magnetic resonance (1H NMR)gel permeation chromatography (GPC) and differentialscanning calorimetry (DSC) analyses Moreover the shortIKVAV peptide was chemically bonded using functionalmonomers on the copolymer film containing carboxyl groupin the side chain The effect of short IKVAV peptide on PC12cell behavior was also determined

2 Materials and Methods

21 Materials Benzyl alcohol (C6H5CH2OH) ethyl alco-

hol (CH3CH2OH) ether (CH

3CH2OCH3CH2) ethyl ace-

tate (CH3COOC

2H5) dodecanol (C

12H25OH) pyridine

(C5H5N) sodium bicarbonate (NaHCO

3) and sodium

nitrite (NaNO2) were purchased from Guangzhou Chemical

Reagent Factory (China) l-Aspartic acid was obtained fromShanghai GiAo Biochemical Co Ltd (China) Bromoace-tyl chloride N-hydroxysuccinimide (NHS) and NN1015840-dicy-clohexylcarbodiimide (DCC) were purchased from AcrosOrganics Triethylamine fromGuangzhou Chemical ReagentFactory was distilled from CaH

2 NN-Dimethylformamide

(DMF) was distilled from 4 A molecular sieve l-LA wasobtained from Huizhou Foryou Medical Device Co Ltd(Guangdong China) and recrystallized three times in ethylacetate Stannous octoate [Sn(Oct)

2] was obtained from Alfa

Aesar Trifluoroacetic acid was purchased from AldrichHydrogen bromide (HBr) in acetic acid (33) was purchasedfrom Acros Organics IKVAV peptides were synthesized byGL Biochem Co Ltd

22 Characterization The mole ratio of l-LA to BMD wasanalyzed by 1H NMR spectroscopy with CDCl

3as solvent

and tetramethylsilane as internal standard Fourier transforminfrared (FTIR) spectra were obtained by NicoletNexus 670Number-average (119872

119899) molecular weights were determined

using GPC (Waters 410 Milford MA) equipped with organicGPC columns Chloroform was used as mobile phase at aflow rate of 10mLmin at 35∘C Thermal properties wereexamined by DSC and the samples were heated from 0∘Cto 200∘C at a heating rate of 10∘Cmin under nitrogenatmosphere Cell morphology was studied using scanningelectron microscopy (SEM JSM-6380LA Analytical JEOLLtd Tokyo Japan) equippedwith vacuummode and at 15 kVimaging

23 Copolymer Synthesis

231 Copolymerization of l-LA with BMD BMD was syn-thesized using l-aspartic acid through a four-step reactionDifferent molar ratios of l-LA and BMD (982 955 and928) were added to a dry Schlenk tube under dry Ar atmo-sphere Dodecanol was added as initiator ([119872][119868] = 7001)with Sn(Oct)

2as catalyst ([119872][119868] = 10001) The tube was

evacuated for 2 h at ambient temperature sealed immersedin an oil bath and heated at 110∘C for 48 h The three typesof copolymers were dissolved in chloroform and precipitatedwith methanol (Scheme 1)

232 Deprotection of Copolymers to Obtain Carboxyl-Sub-stituted Polymers Copolymers with protecting groups (2 g)were dissolved in trifluoroacetic acid (20mL) Briefly 33HBrCH

3COOH solution (8mL) was added dropwise under

Ar atmosphere after the complete dissolution of the copoly-mer The product was poured into anhydrous ether (200mL)after 5 h at ambient temperature The precipitates were dis-solved in chloroform and purified by ethanol

24 Modification of Copolymer Films by Short Peptides Co-polymers after deprotection (2 g) were dissolved in chlo-roform (30mL) NHS (3mmol 06 g) and DCC (3mmol034 g) were added dropwise The compounds were reactedfor 2 h in an ice bath and for 24 h at room temperature Theproduct was dissolved in ethanol and precipitated withchloroform

The polymer films were prepared by solution pouringand solvent evaporationThe samples were then immersed in10mgmL short-peptide solution after drying The film wasthen soaked for 12 h with mild agitation and washed threetimes (once per hour) with phosphate-buffered saline (PBS)solution to remove unreacted short peptides

25 Cell Culture and Morphology Observation The polymerfilm functionalized with peptide on 15mm cover slip wasplaced in a 24-well plate and pressed with ring to ensure com-plete contact of the scaffolds to the wellsThe specimens weresterilized under UV light washed three times with PBS andimmersed in Dulbeccorsquos modified Eaglersquos medium (DMEM)overnight before cell seeding Rat pheochromocytoma-derived cell line (PC12) was seeded on the film at a densityof 10 times 104 cellswell and cultured in DMEM containing


CH3

120575 (ppm)8 7 6 5 4 3 2 1

10 09 08

(h + c) (g)

(f) (e) (d)

(b)

O O OO O

O

O

O

m

O

O O

Hn

(a)

(b)

(c)

(d)

(e) (f) (h)

(g)

(h)

(g)

(a)

CH3

H3C(H2C)10H2C



3)



119899

c (gmol) 119872119899

c (gmol) 119872119908119872119899

c 119879119892

d (∘C) 119879119898

d (∘C) Δ119867119898












3500 3000 2500 2000 1500 1000 500

(a)

(b)



1728 cmminus1


10 11 12 13 14 15 16 17

(c)(b)

Elution time (min)

(a)


98-co-BMD

2) (b) P(LA

95-co-BMD

5) and

(c) P(LA92-co-BMD

8)



98-co-BMD

2) P(LA

95-

co-BMD5) and P(LA

92-co-BMD



119899decreased and


0 50 100 150

(c)

(b)

Endo (a)

T (∘C)


98-co-BMD

2) (b) P(LA

95-co-

BMD5) and (c) P(LA

92-co-BMD

8)


119898 119879119892



98-


95-

co-BMD5) and P(LA

92-co-BMD



BMD5) and P(LA

92-co-BMD



4 Conclusions






95-

co-BMD5) and P(LA

92-co-BMD





Competing Interests


Acknowledgments


References

































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




CH3

120575 (ppm)8 7 6 5 4 3 2 1

10 09 08

(h + c) (g)

(f) (e) (d)

(b)

O O OO O

O

O

O

m

O

O O

Hn

(a)

(b)

(c)

(d)

(e) (f) (h)

(g)

(h)

(g)

(a)

CH3

H3C(H2C)10H2C



3)



119899

c (gmol) 119872119899

c (gmol) 119872119908119872119899

c 119879119892

d (∘C) 119879119898

d (∘C) Δ119867119898












3500 3000 2500 2000 1500 1000 500

(a)

(b)



1728 cmminus1


10 11 12 13 14 15 16 17

(c)(b)

Elution time (min)

(a)


98-co-BMD

2) (b) P(LA

95-co-BMD

5) and

(c) P(LA92-co-BMD

8)



98-co-BMD

2) P(LA

95-

co-BMD5) and P(LA

92-co-BMD



119899decreased and


0 50 100 150

(c)

(b)

Endo (a)

T (∘C)


98-co-BMD

2) (b) P(LA

95-co-

BMD5) and (c) P(LA

92-co-BMD

8)


119898 119879119892



98-


95-

co-BMD5) and P(LA

92-co-BMD



BMD5) and P(LA

92-co-BMD



4 Conclusions






95-

co-BMD5) and P(LA

92-co-BMD





Competing Interests


Acknowledgments


References

































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




3500 3000 2500 2000 1500 1000 500

(a)

(b)



1728 cmminus1


10 11 12 13 14 15 16 17

(c)(b)

Elution time (min)

(a)


98-co-BMD

2) (b) P(LA

95-co-BMD

5) and

(c) P(LA92-co-BMD

8)



98-co-BMD

2) P(LA

95-

co-BMD5) and P(LA

92-co-BMD



119899decreased and


0 50 100 150

(c)

(b)

Endo (a)

T (∘C)


98-co-BMD

2) (b) P(LA

95-co-

BMD5) and (c) P(LA

92-co-BMD

8)


119898 119879119892



98-


95-

co-BMD5) and P(LA

92-co-BMD



BMD5) and P(LA

92-co-BMD



4 Conclusions






95-

co-BMD5) and P(LA

92-co-BMD





Competing Interests


Acknowledgments


References

































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







95-

co-BMD5) and P(LA

92-co-BMD





Competing Interests


Acknowledgments


References

































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials

















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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