DTIC ADA630574: Sequential Delivery of BMP-2 and IGF-1 Using a Chitosan Gel with gelatin Microspheres Enhances Early osteoblastic Differentiation

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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
01 MAY 2012 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin
5b. GRANT NUMBER
microspheres enhances early osteoblastic differentiation
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Kim S., Kang Y., Krueger C. A., Sen M., Holcomb J. B., Chen D., Wenke
5e. TASK NUMBER
J. C., Yang Y.,
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United Stataes Army Institute of Surgical Research, JBSA Fort Sam REPORT NUMBER
Houston, TX
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14. ABSTRACT
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S.Kimetal./ActaBiomaterialia8(2012)1768–1777 1769
(PDLFs)[11].Intheirexperiments,thereleaseofgrowthfactorswas anaqueousethanolsolutiontoremove theresidualcross linking
controlled by BMP 2 containing basic gelatin microspheres and agentontheirsurfacesandthenfreeze driedovernight.Theywere
IGF 1containingacidicgelatinmicrospheres,whichwereincorpo thensievedtoobtainparticlesrangingfrom50to100lm.
ratedintoglycidylmethacrylateddextran(Dex GMA)scaffolds.
We have previously developed a thermosensitive injectable 2.3.1.Fouriertransforminfraredspectroscopy(FTIR)spectra
chitosangeltodeliverBMP 2.Ithasbeenfoundtosignificantlyen In order to investigate chemical structure of both gelatin MSs
hancetheosteoblasticdifferentiationofmouseosteoblastprecur and cross linkedgelatin MSs, FTIR spectrawere obtained using a
sor cells and the mineralization of human embryonic palatal Nicolet FTIR infrared microscope coupled to a PC with analysis
mesenchymalcells[25].Chitosangelshavebeenusedduetotheir software.SampleswereplacedintheholderdirectlyintheIRlaser
excellent biocompatibility, enzyme regulated degradation, and beam.Allspectrawererecordedbytransmittancemode(100times
highefficacyofdrugtherapy[2,3,9,25 28].Additionally,therapeu scanning,650 4000cm 1).
tic agents are released via the diffusion or biodegradation of the
chitosanpolymers[5,25 28].Thepurposeofthisstudywastocre 2.3.2.Degreeofcross linking
ate and characterize a sequential delivery system consisting of a Degree of cross linking of the gelatin MSs was determined by
chitosangelandgelatinmicrospheres(MSs)toachieveasequen ninhydrin assay, which was used to determine the percentage of
tial release of BMP 2 and IGF 1. We hypothesized that an initial freeaminogroupsremaininginthegelatinMSsaftercross linking.
releaseofBMP 2fromthechitosangelfollowedbythereleaseof Thecross linkedgelatinMSswerepreparedwithdifferentconcen
IGF 1 from the gelatin MSs would enhance osteoblastic activity trations of glyoxal (10, 20, 50, or 100mM). The samples were
of bone cells. In this study, we made glyoxal cross linked gelatin heatedintheninhydrinsolutionat100(cid:3)Cfor10min,andthelight
MSsfordeliveryofIGF 1,whichwerethenencapsulatedintothe absorbanceat550nmwasrecordedusingamicroplatereader(TE
chitosan gel formulation. Furthermore, we aimed to characterize CAN Infinite F50). Glycine (Fisher Scientific, Fair Lawn, NJ) was
thedegreeofcross linking,degradation,releaserateandcytotox usedasanaminoacidnitrogenstandardatvariousknownconcen
icityofthedeliverysystem.Wealsoevaluatedosteoblasticactivity trations.Thedegreeofcross linking(D)ofthesampleswascalcu
c
by measuring ALP specific activity of preosteoblast W 20 17 latedfollowingtheequationD =[(A B)/A](cid:3)100,whereAismole
c
mousebonemarrowstromalcells. fractionoffreeaminogroupinun cross linkedgelatinMSsandBis
molefractionoffreeaminogroupincross linkedgelatinMSs.
2.Materialsandmethods
2.3.3.SwellingofgelatinMSsatdifferenttemperatures
Toevaluatetheeffectofcross linkingonwaterstabilityofthe
2.1.Materials
gelatin MSs, the swelling characteristic of the gelatin MSs was
investigatedatdifferenttemperatures.Theswellingofthegelatin
Chitosan (P310kDa, 75% or greater degree of deacetylation),
MSs and the cross linked gelatin MSs (50mM) were observed
disodium b GP (glycerol 2 phosphate disodium salt hydrate; cell
usingamicroscope(NikonECLIPSETE 2000 U).Thedriedsamples
culture grade), and glyoxal (40wt.%) were purchased from Sig
wereplacedintoacontainerwithPBS(pH7.4)andincubatedat4
ma Aldrich (St Louis, MO). Gelatin type B, olive oil, acetone, and
or37(cid:3)Cfor3days.PhotomicrographsofthegelatinMSswerepro
ethanolwereall purchasedfromFisher Scientific(FairLawn, NJ).
cessed at 6h, 1day, and 3days of incubation using MetaVue
Allotherchemicalswerereagentgradeandwereusedasreceived.
software.
Humanbonemorphogeneticprotein 2(BMP 2)wasobtainedfrom
PeproTech (Rocky Hill, NJ) and recombinant human insulin like
2.3.4.Cytotoxicity
growthfactorI(IGF 1)waspurchasedfromR&DSystems(Minne
W 20 17 cells were grown and maintained in DMEM media
apolis,MN).Fetalbovineserum(FBS),Trypsin EDTA,L glutamine,
antibiotic antimycotic,phosphate bufferedsaline(PBS),andDul with10%FBS,1%antibiotic/antimycoticmixture,5mlL glutamine
(200mM),andsodiumpyruvate.Thiscelllinehasbeenusedinan
becco’smodifiedEagle’smedium(DMEM)wereallpurchasedfrom
ASTMF2131toevaluateactivityofBMP 2invitro.Cellculturewas
Invitrogen™ (Eugene, OR). W 20 17 cells were cultured as per
achievedinanincubatorsuppliedwith5%CO at37(cid:3)C.Theculture
AmericanTypeCultureCollection(ATCC)instructions. 2
medium was changed every 3days. In order to investigate the
cytotoxicity of the gelatin MSs, the W 20 17 cells were cultured
2.2.Preparationofgelatinmicrospheres intheDMEMmediacontainingthegelatinMSs.Cellswereseeded
in24 wellplatesatadensityof30,000cellsperwellandincubated
GelatinMSswerepreparedusingawater in oilemulsiontech with10mgofthegelatinMSsfor3days.Afterincubationof1and
nique.Briefly,agelatinsolutionwaspreparedbydissolving1ggel 3days, the number of viable cells was determined quantitatively
atinpowderin10mldistilledwaterat50(cid:3)C.Thesolutionwasthen usingaCellTiter96AQueousOneSolution(MTS)assayaccording
added dropwise to 60ml olive oil, which was preheated to 50(cid:3)C to the manufacturer’s instructions. Before the assay, the cellular
whilestirringat500rpmusingastraight bladeimpeller.Thegel morphologywasobservedqualitativelyusingamicroscope(Nikon,
atin solution was allowed to emulsify for 10min. Subsequently, ECLIPSE TE 2000 U). Photomicrographs of cells were processed
theentireemulsificationbathwaschilledto4(cid:3)Conicewithcon usingNikonMetaVuesoftware.
tinuous stirring at 500rpm for 40min, and gelatin MSs were
formed. The gelatin MSs were collected by filtration and washed 2.4.GelatinMSsencapsulatedchitosangelcomposites
with chilled acetone and ethanol. Finally, the obtained gelatin
MSswerefreeze driedovernight. A 1.5% (w/v) chitosan solution was prepared by stirring pow
deredchitosanin0.75%(v/v)aqueousaceticacidatroomtemper
2.3.Cross linkingofgelatinMSs ature overnight. The insoluble particles in the chitosan solution
were removed by filtration. A 50% (w/v) b GP solution was pre
ThepreparedgelatinMSsweredispersedintoanaqueouseth paredindistilledwaterandsterilizedusingPESsyringefilterswith
anol solution containing different concentrations of glyoxal (10, 0.22lm pore size (MillexTM, MA) and stored at 4(cid:3)C. 50ml of
20,50,or100mM)andstirredatroomtemperatureforcross link chitosan solution was dialyzed at room temperature against 1l
ingfor10h.Thecross linkedgelatinMSswererinsedtwicewith of distilled water for 7days with daily changes of water (1l) in
1770 S.Kimetal./ActaBiomaterialia8(2012)1768–1777
an8kDacutoffdialysismembranetoreducetheaceticacidcon amountsofIGF 1fromthematerialsweredeterminedasafunction
tent.ThefinalpHvalueofthechitosansolutionwas6.3.Thedia oftimebyanIGF 1ELISAkit(RayBio,GA).Briefly,100lloftheob
lyzed chitosan solution was autoclaved at 121(cid:3)C for 20min, tained samples were pipetted into a 96 well IGF 1 microplate
cooleddowntoroomtemperature, andstoredat4(cid:3)C. Thecross coated with anti human IGF 1 and incubated at 4(cid:3)C overnight.
linkedgelatinMSswerethenencapsulatedintothechitosansolu After washing each well with wash buffer provided by the ELISA
tiononiceandvortexed.Sterilized,ice coldb GPsolution(2.31M) kit for a total of four washes, 100ll of biotinylated anti human
was added drop by drop to the chitosan solution under stirring IGF 1wasaddedtoeachwellandincubatedatroomtemperature
conditions in an ice bath. The final concentration of b GP in the for1h.Afterrepeatingthewashingstep,eachwellwasfilledwith
chitosansolutionwas88mM,andthefinalpHvalueofthechito 100ll of horseradish peroxidase streptavidin solution and incu
san gel formulation was 7.2. Each gel forming solution was al bated at room temperature for 45min. After the washing step,
lowedtocompletelybecomeagelinanincubatorfor3hat37(cid:3)C. 100llofTMB(3,30,5,50 tetramethylbenzidine)wasaddedtoeach
wellandincubatedfor30minatroomtemperatureinthedark.Fi
2.5.Dissolutionrate nally,50llofstopsolutionwasaddedintoeachwell.Theoptical
densityofeachwellwasdeterminedusingamicroplatereaderat
Gelatin is an amphoteric protein containing both positively 450nm(TECANInfiniteF50).
chargedandnegativelychargedaminoacids.Iteasilydissolvesin
water at body temperature, releasing amino acids. In this study, 2.7.2.BMP 2release
thecross linkedgelatinMSsorthecross linkedgelatinMS loaded The in vitro BMP 2 release profile from the chitosan gel was
chitosangelwereplacedinacontainercontaining2mlofPBS(pH investigated for 1week. BMP 2 solution was added directly into
7.4) and incubated at 37(cid:3)C for 5days. At predetermined time thechitosansolutiononiceandvortexed.88mMofcoldb GPsolu
points,500llaliquotsofthemediumweresampledandthesame tionwasaddedintothemixturetocompletethegel formingsolu
amountoffreshPBS(pH7.4)wasaddedintoeachcontainer.Inthe tion.Eachgel formingsolutioncontainingBMP 2wasallowedto
collectedfractions, thecumulative amountsof dissolved proteins completely become a gel in an incubator at 37(cid:3)C. Eventually,
from the gelatin MSs or the combination were determined as a 50ngml–1ofBMP 2waspresentwithineachsample(BMP 2(Gel)).
function of time by bicinchoninic acid (BCA) assay (Pierce, Rock BMP 2 (Gel) was placed in a container containing2ml of PBS
ford,IL).Theopticaldensityofeachsamplewasdeterminedusing (pH 7.4) and incubated at 37(cid:3)C for a week. At designated time
amicroplatereaderat562nm(TECANInfiniteF50). points, 300ll aliquots of the release medium were sampled and
thesameamountoffreshPBS(pH7.4)wasaddedintoeachcon
2.6.Scanningelectronmicroscopy(SEM) tainer.In the collected fractions, the cumulative release amounts
of BMP 2 from the chitosan gels were determined as a function
Thesurfacemorphologyofthematerialswasobservedtoexam oftimebyaBMP 2ELISAkit(R&Dsystems,MN).Briefly,50llof
inecompatibilityofgelatinMSswithachitosangelafterimplanta the obtained supernatant was pipetted into a 96 well BMP 2
tionatbodytemperature.Threedifferentmaterials,i.e.achitosan microplate coated with a mouse monoclonal antibody and incu
gel,anun cross linkedgelatinMS loadedchitosangel,andacross batedfor2hatroomtemperature.Afterwashingeachwellwith
linked gelatin MS loaded chitosan gel, were prepared. Theywere wash buffer provided by the ELISA kit for a total of four washes,
incubated at 37(cid:3)C for 5h and lyophilized overnight (Freezone, 200llofBMP 2conjugatewasaddedtoeachwellandincubated
LABCONCO). The samples were sputter coated with gold and atroomtemperaturefor2h.Afterrepeatingthewashingstep,each
examined under a scanning electron microscope (FEI, USA) oper well was filled with 200ll of BMP 2 substrate and incubated at
atedat15kV. room temperature for 30min in the dark. Finally, 50ll of stop
solutionwasaddedintoeachwell.Theopticaldensityofeachwell
2.7.Invitroreleasestudies wasdeterminedusingamicroplatereaderat450nmwitha cor
rectionsettingof540nm(TECANInfiniteF50).
2.7.1.IGF 1release
InvitroIGF 1releaseprofilesfromcross linkedgelatinMSsora 2.8.Invitroanalysis
cross linked gelatin MS loaded chitosan gel were examined for
1week. IGF 1 loading was achieved by a method of adsorption. 2.8.1.EffectofgrowthfactorsonALPspecificactivityofW 20 17
Thecross linked gelatinMSswereloadedwithIGF 1byswelling Tobetterevaluatethesequentialdeliveryofgrowthfactorson
in aqueous IGF 1 solutions (IGF 1(MSs)). IGF 1 (isoelectric point the cell responses, we first established a growth factor cell re
(IEP)=8.6)ispositivelycharged,andtherefore,negativelycharged sponsecalibrationmodel.WestudiedALPactivityasanindicator
typeBgelatinformsapolyioniccomplexationwithIGF 1[23,29 ofearlyosteoblasticdifferentiationtodesignatedsingularorcom
31]. IGF 1 solution was dripped onto the microparticles at a vol binationofBMP 2andIGF 1listedinTable1.Inthisexperiment,
umeof25llpermgofthecross linkedgelatinMSs.Theresulting W 20 17 cells were treated with growth factors (BMP 2, IGF 1,
mixturewasvortexedandincubatedat4(cid:3)Cfor10hbeforefreeze or combinations), and ALP activity and double stranded DNA
drying. Eventually, 50ngml–1 of IGF 1 was present within each (dsDNA)ofW 20 17cellsweredetermined.Thecellswereseeded
sample (IGF 1(MSs)). The IGF 1 loaded gelatin MSs were then in24 wellplatesatadensityof30,000cellsperwellandcultured
encapsulated into a chitosan gel formulation (IGF 1(gel+MSs)). for 7days. On days 1 and 3, 50ngml–1 of each growth factor or
TheIGF 1loadedmicroparticleswereaddedintothechitosangel their combination was added into the culture medium as shown
formulation on ice and vortexed. 88mM of cold b GP solution inTable1.Theculturemediumwaschangedevery3days.
wasaddedintothemixturetocompletethegel formingsolution. At designatedtime points (5 and 7days) the mediumwas re
Eachgel formingsolutionwasallowedtocompletelybecomeagel moved from the cell culture. The cell layers were washed twice
inanincubatorat37(cid:3)C. withPBS (pH 7.4)and thenlysed with1ml of 0.2%Triton X 100
IGF 1(MSs)orIGF 1(gel+MSs)was placedinacontainer con and three freeze thaw cycles, which consisted of freezing at
taining2mlofPBS(pH7.4)andincubatedat37(cid:3)Cforaweek.At 80(cid:3)C for 30min immediately followed by thawing at 37(cid:3)C for
designatedtimepoints,300llaliquotsofthereleasemediumwere 15min.50llaliquotsofthecelllysatesweresampledandadded
sampledandthesameamountoffreshPBS(pH7.4)wasaddedinto to50llofworkingreagentina96 wellassayplate.Theworking
each container. In the collected fractions, the cumulative release reagent contains equal parts (1:1:1) of 1.5M 2 amino 2 methyl

1776 S.Kimetal./ActaBiomaterialia8(2012)1768–1777
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