tures田中丽奈

Anovel,visiblelight-induced,rapidlycross-linkablegelatinscaffoldforosteochondraltissueengineeringTetsuroMazaki1,2,YasuyukiShiozaki1,2,KentaroYamane1,2,AkiYoshida1,MarikoNakamura3,YasuhiroYoshida4,DiZhou5,TakashiKitajima5,MasatoTanaka1,YoshihiroIto5,ToshifumiOzaki1*&AkihiroMatsukawa2*1DepartmentofOrthopaedicSurgery,GraduateSchoolofMedical,DentistryandPharmaceuticalSciences,OkayamaUniversity,2-5-1Shikata,Okayama700-8558,Japan,2DepartmentofPathologyandExperimentalMedicine,GraduateSchoolofMedical,DentistryandPharmaceuticalSciences,OkayamaUniversity,2-5-1Shikata,Okayama700-8558,Japan,3DepartmentofHealthandWelfareProgram,KibiInternationalUniversityJuniorCollege,8Iga-machi,Takahashi,Okayama716-8508,Japan,4DepartmentofBiomaterials,GraduateSchoolofMedical,DentistryandPharmaceuticalSciences,OkayamaUniversity,2-5-1Shikata,Okayama700-8558,Japan,5NanoMedicalEngineeringLaboratory,RIKEN,2-1Hirosawa,Wako351-0198,Japan.
Osteochondralinjuriesremaindifficulttorepair.
Wedevelopedanovelphoto-cross-linkablefurfurylamine-conjugatedgelatin(gelatin-FA).
Gelatin-FAwasrapidlycross-linkedbyvisiblelightwithRoseBengal,alightsensitizer,andwaskeptgelledfor3weekssubmergedinsalineat376C.
Whenbonemarrow-derivedstromalcells(BMSCs)weresuspendedingelatin-FAwith0.
05%RoseBengal,approximately87%ofthecellswereviableinthehydrogelat24hafterphoto-cross-linking,andthechondrogenicdifferentiationofBMSCswasmaintainedforupto3weeks.
BMP4fusionproteinwithacollagenbindingdomain(CBD)wasretainedinthehydrogelsathigherlevelsthanunmodifiedBMP4.
Gelatin-FAwassubsequentlyemployedasascaffoldforBMSCsandCBD-BMP4inarabbitosteochondraldefectmodel.
Inbothcases,thedefectwasrepairedwitharticularcartilage-liketissueandregeneratedsubchondralbone.
Thisnovel,photo-cross-linkablegelatinappearstobeapromisingscaffoldforthetreatmentofosteochondralinjury.
Managementandrepairofosteochondralinjuriesinthekneeremainchallenging.
Osteochondralinjuriesinvolvedamagetoboththearticularcartilageandtheunderlyingsubchondralbone.
Articularcartilage(hyalinecartilage)haslimitedintrinsichealingcapabilities.
Onceinjured,lesionsaretypicallyreplacedbyfibrocartilage,ascartissuewithdifferentbiomechanicalpropertiesandimpairedmechanicalfunction1,2.
Coveringtheload-bearingsurfacesofjointboneswithfibrocartilagefailstoprotectthesubchondralbonefromfurtherdegeneration.
Asosteochondraldefectsareoftenassociatedwithjointmechanicalinstability,thepotentialproblemsafterosteochondralinjuryincludeearlyosteoarthritisand/orosteoarthrosis(OA)3.
Thegoalsfortreatmentofosteochondralinjuriesaretorestorehyalinecartilageandsubchondralboneregeneration.
Overthelastfewdecades,progresshasbeenmadeusingsurgicalrepairinterventions.
Recentadvancesinthesetreatmentmethodsincludetheuseoftissuespecificcells,progenitorcells,andappropriatecellularstimulationwithgrowthfactorsattheaffectedsites4,5.
Currently,tissue-engineeringapproachesusingscaffold-basedproce-dureshavebeenattractingincreasingattention6.
Ascaffoldprovidesanenvironmentforcellattachment,pro-liferation,anddifferentiation.
Inaddition,scaffoldscanbeusedtoachievedrugdeliverywithhigh-loadingefficiencyatspecificsites.
Biologicscaffoldsemployedtodateasnaturalpolymersincludealginate,collagen,fibrin,albumin,hyaluronan,platelet-richplasma,andgelatin7.
Gelatinisaverypromisingmaterialforcellproliferationandtissueregeneration,buthasthedisadvantageofmechanicalweakness.
Recently,chemicalmodificationofgelatinhasbeenreportedtoimproveitsmechanicalpropertiesbycross-linkingwithvisiblelight8–12.
Photo-inducedcross-linkingorpolymerizationisafastandconvenientwaytoproducegelsorhigh-molecular-weightpolymers.
Thescaffoldcanbeformedatspecificsitesusinginjectablesolutionsandvisiblelight,makingtheapplicationtoadesignatedsitesimpleandminimallyinvasive.
Wehavepreviouslydevelopedanewtypeofgelatinbyincorporatingfurfurylisocyanate(gelatin-FI),OPENSUBJECTAREAS:PRE-CLINICALSTUDIESBIOMEDICALMATERIALSReceived11November2013Accepted6March2014Published25March2014CorrespondenceandrequestsformaterialsshouldbeaddressedtoA.
M.
(amatsu@md.
okayama-u.
ac.
jp)*Theseauthorscontributedequallytothiswork.
SCIENTIFICREPORTS|4:4457|DOI:10.
1038/srep044571whichwasphoto-cross-linkedwithRoseBengal(RB),afooddye.
Thephoto-crosslinkablegelatin-FIwasusefulasadirectpulpcap-pingmaterialinthedentalfield13.
Tissuecellsfeelandrespondtothestiffnessoftheirsubstrate14.
Thephysicalpropertiesofscaffoldinfluencecellfunctionandtissuemorphogenesis15.
Consequently,physicalcharacteristicsmustbeconsideredwhendesigninghydrogelsfortissue-engineeringapplica-tions16.
Itwasfoundthatthestiffnessofthehydrogelstronglyaffec-tedthecellattachment,focaladhesion,migrationandproliferation17.
Here,wedevelopedanothernewgelatinderivativethatwasmodifiedwithfurfurylamine(gelatin-FA).
Gelatin-FAwasconsolidatedbyvisiblelightmorerapidlywithstifferpropertiesthangelatin-FIinthepresenceofRB.
Inthepresentstudy,wedemonstratethatthisnovelmodifiedgelatin-FAisaneffectivescaffoldforosteochondralrepairinarabbitosteochondraldefectmodel.
Gelatin-FAhydrogelswereusedtodeliverbonemarrowstromalcells(BMSCs)andbonemorphogeneticprotein-4(BMP4)withcollagenbindingdomains(CBD)totheaffectedsites.
Theresultssuggestthatthismodifiedgelatin,incombinationwithcellsandcollagen-bindinggrowthfac-tors,isapromisingscaffoldfortissueengineeringbecauseuniformcellseedingiseasytoachieve;theimplantedcellssurviveanddiffer-entiate;andgrowthfactor(s)withCBDareretainedinthehydrogel.
ResultsPropertiesofgelatin-FA.
Aftervisiblelightirradiation,themixturewastransformedfromsolutiontogelbyaphoto-oxidationcross-linking(POC)mechanism.
Thechemistryofgelatin-FAisshowninFig.
1a.
Byutilizingthecarboxygroupsinthegelatinforcouplingwithfuran,theremainedaminogroupsinthegelatinwereexpectedtocontributetheadhesivenessandstiffnessaftercrosslinking.
The1H-NMRspectraofgelatinbeforeandafterfurfurylamineconju-gationareshowninFigure1b.
Inunconjugatedgelatin,therewasabroadpeakat7.
25ppm,contributedmostlybythebenzeneprotonofphenylalanineresidues.
Inadditiontothispeak,gelatin-FAandgelatin-FIhadthreenewpeaksfoundat6.
1,6.
2,and7.
3ppm,attributedtothefurangroup.
Theapproximatecompositionofaromaticaminoacidsinporcinegelatinis2%,whichissimilartotheestimateforfurangroupsingelatin-FA(2.
9%)basedonthepeakareain1H-NMRspectra.
Tocomparegelatin-FAwithourpreviouslyreportedgelatin-FI13,10%aqueoussolutionsofgelatin-FAandgelatin-FIweremixedwithRB(0.
5%)onaplateandilluminatedfor10,30,60,and180sbyvisiblelight.
Theplatewaswashedtoremoveuncross-linkedsolutionsandthegelationratewasobserved.
AsshowninFigure1c,approximately75%ofgelatin-FAformedahydrogelafter30sofillumination.
Conversely,gelatin-FIremainedasolutionafter60sofillumination,andonly15%wasgelledafter180sofillumination,indicatingthatgelatin-FAhasmorerapidcross-linking.
Next,therheologicalpropertiesofthemodifiedgelatinwereexaminedafterilluminationwithvisiblelight.
Boththestorage(G9)andloss(G0)moduliofgelatin-FAhydrogelswerehigherthanthemoduliofgelatin-FIhydrogels,suggestingthatgelatin-FAhydro-gelshadhigherelasticityandflexibility(Fig.
2).
IthasdemonstratedthathydrogelwithhigherG9showedsignificantlyup-regulatedexpressionsofosteocalcinandrunt-relatedtranscriptionfactor2(RUNX2),withthepresenceofalkalinephosphatase,andtheevid-enceofcalciumaccumulation15.
Meanwhile,theG0/G9(equivalenttotand)ratioofgelatin-FAhydrogelswasrelativelylow,indicatingaFigure1|Propertiesofmodifiedgelatin.
(a)Syntheticschemeofmodifiedgelatinandthecross-linkingmechanism.
(b)1H-NMRspectraofgelatininD2Obeforeandaftermodification.
(c)Timecourseofgelformationoffurfuryl-conjugatedgelatin(gelatin-FAandgelatin-FI,each10%)withRoseBengal(0.
5%)aftervisiblelightillumination.
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com/scientificreportsSCIENTIFICREPORTS|4:4457|DOI:10.
1038/srep044572lowviscositysimilartogelatin-FIhydrogels(Fig.
2).
Thesedatasuggestthatgelatin-FAhasbetterappropriatephysicalpropertiesrelativetogelatin-FI.
Cytotoxicityandphototoxicity.
TheRBcytotoxicityandphototo-xicitytowardBMSCswereinvestigated.
Thedataassessedat24hwereshowninFigure3a.
RBwascytotoxictomorethan70%ofthecellsat1%RB.
At0.
1%RB,thepercentageofviablecellswassignificantlyimprovedinthepresenceofgelatin-FA,indicatingacytoprotectionfunctionofgelatin-FA.
Gelatin-FAalsoreducedthephototoxicitybyvisiblelightat0.
1%and0.
01%RBconcentrations(Fig.
3a).
BMSCsingelatin-FAwerekeptaliveatthesamelevelfor7daysat0.
01%RBwithorwithoutlightillumination.
EvenatahigherconcentrationofRB(0.
1%),about50%BMSCswerealiveingelatin-FAafter7dayswhereasallofthecellsweredeadwithoutgelatin-FA(Fig.
3b).
Next,thephoto-cross-linkableintensitywasexamined.
Todothis,gelatin-FA(15%)withvariousconcentrationsofRB(0.
005,0.
01,0.
05,0.
1%)wassubmergedinPBS,andexposedtovisiblelightfor2min.
Gelatin-FAhydrogelswith0.
05%RBweremacrosco-picallyunchangedinPBSforatleast21daysat37uC(Fig.
3c).
RBconcentrationsbelow0.
01%failedtoformsolidgelsinthesubmergedconditions(notshown).
Theviabilityofcellsingelatin-FAhydrogelswith0.
05%RBwas87.
3%onday1,55.
8%onday3and44.
1%onday7afterlightexposureinthesubmergedconditions(Fig.
3d).
Thepercentviablecellsat24hin0.
05%RBwaslargerthanthosein0.
1%RB(64.
0%viable,Fig.
3a).
Thus,0.
05%RBwaschosenasasuitableconcentrationforcell-basedscaffolds.
WhenBMSCswereculturedinthegelatin-FAhydrogels,DNAandacidicmucopolysaccharidecontentsweremeasured.
AlthoughtheDNAcontentinthehydrogelsdecreasedwithtime,theacidicmucopoly-saccharidelevelwasunchangedthroughouttheobservationperiod.
Theresultingacidicmucopolysaccharide-to-DNAratio,reflectingchondrogenicdifferentiationofBMSCs,increasedwithtimeforupto3weeks,suggestingthatBMSCsdifferentiatedintochondrocytesinthegelatin-FAscaffold(Fig.
3e).
Gelatin-FAasascaffoldforBMSCimplantation.
Toexaminewhethergelatin-FAcouldbeusefulfortissueengineering,wefirstemployeditasascaffoldforBMSCs.
BMSCs(1.
53105cells)weresuspendedin15%gelation-FAcontaining0.
05%RB,andthemix-tureswereimplantedintorabbitosteochondraldefectsandexposedtovisiblelightfor2min(Fig.
4a).
Fourweekslater,thedefectlesionwasfilledwithregeneratedtissues(Fig.
4b).
Nomeaningfuldiffe-renceswerefoundingelatin-FA-treatedgroupsascomparedwiththeuntreatedcontrolsatthistimepoint.
At12weeks,thedefectlesionshoweddegenerativechangesintheuntreatedgroup.
Regene-rativetissuefilledthedefectinthegelatin-FAgroup,althoughthesurfaceappearedroughened.
Inthegelatin-FAplusBMSCsgroup,thearticularsurfaceappearedtobesimilartothesurroundingintactcartilage(Fig.
4b).
Thegrossgradingscoredemonstratedanimprovementindefectsafterthetreatmentwithgelatin-FAplusBMSCs.
Gelatin-FAbyitselfalsoimprovedtherepair(Fig.
4c).
Histologically,untreatedosteochondraldefectswerenotcoveredwithregenerativetissues.
Gelatin-FA-treatmentallowedgranula-tiontissuetofillthedefectat4weeks(Fig.
4d).
Macrophageswereevidentinthegranulationtissueingelatin-FA-treatment,whichweremoreprominentingelatin-FAplusBMSCs(Fig.
4e).
Hyalinecartilage-likecellswereseenat12weekswiththeirsurroundingmatrixstainedwithsafraninO(Fig.
4d).
Thestainedareainrepairedlesionswassignificantlylargerinthegelatin-FAplusBMSCsgroupascomparedwiththegelatin-FAgroup(Table1).
Histologicalgradingscoresat4and12weeksaftersurgeryre-vealedthattreatmentwithgelatin-FAplusBMSCsimprovedtissueregeneration(Fig.
4f).
Thescoreat12weekswassignificantlyhigherthanat4weeks(P,0.
01,byMann–WhitneyUtest).
Gelatin-FAwithoutBMSCsalsoshowedanimprovedrepaircomparedwithuntreatedcontrols(Fig.
4f).
TypeIIcollagen,notfoundinuntreatedgroups,waspresentinthegelatin-FAaloneandgelatin-FAplusBMSCsgroups,andthestainingwasmoreintenseinthegelatin-FAplusBMSCsgroup(Fig.
4g).
Likewise,aggrecanwasnotfoundinuntreatedgroupsbutstainedingelatin-FAplusBMSCsFigure2|Storage(G9)andloss(G0)moduliofgelatin-FAandgelatin-FIhydrogels(10%)at376Ccross-linkedbyvisiblelightinthepresenceofRoseBengal(0.
5%).
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com/scientificreportsSCIENTIFICREPORTS|4:4457|DOI:10.
1038/srep044573group(Fig.
4h).
Subchondralbonerepairwasfoundinthegelatin-FAplusBMSCsgroup(Fig.
5a).
TheBV/TVvaluewassignificantlyhigheraftertreatmentwithgelatin-FAplusBMSCsthanintheothergroups(Fig.
5b).
Allthesefindingssupportthismodifiedgelatin-FAasausefulcellscaffoldforthetreatmentofosteo-chondraldefects.
Gelatin-FAasascaffoldforgrowthfactors.
Wethenaskedwhethergelatin-FAcouldbeusedasascaffoldforgrowthfactor-basedtissueengineering.
WeusedBMP4andCBD-BMP4asgrowthfactors.
CBD-BMP4exhibitedstrongerandmorestablecollagenbindingactivitythandidwild-typeBMP413,18.
Becausegelatinisahydro-lyzedformofcollagen,wehypothesizedthatCBD-BMP4maybebetterretainedingelatin-FAhydrogelsthannativeBMP4.
Totestthis,BMP4andCBD-BMP4(380nM)weresuspendedinseparategelatinsolutions(15%gelatin-FAand0.
05%RB),photo-cross-linkedbyvisiblelight,andthenculturedinPBSfor7days.
ThesupernatantswerecollectedandBMP4concentrationsmeasuredbyELISA.
AsshowninTable2,BMP4wasrapidlyreleasedfromgelatin-FAhydrogels,whereasCBD-BMP4wasretainedinthehydrogelsforatleast7days.
WenextimplantedBMP4-orCBD-BMP4-containinggelatinsolutions(15%gelatin-FAand0.
05%RB)intoosteochondralbonedefectsandexposedthemtovisiblelightfor2min.
Growthfactor-loadedgelatin-FAhydrogelsfacilitatedcartilagerepair(Fig.
6a–d).
Inparticular,CBD-BMP4-loadedgelatin-FAshowedcompletefillingofthedefectwithatissuethatresembledthesurroundingnormalhyal-inecartilageat12weeks(Fig.
6a).
RepairtissueinthedefectstreatedFigure3|Cytotoxicityandphototoxicity.
(a)BMSCs(53103cells)wereculturedingelatin-FA(15%)andvariousconcentrationsofRB(0.
01,0.
1,and1%)andgelledbyilluminationwithvisiblelightfor2min.
After24h,the%cellviabilitywasdetermined.
*P,0.
05,**P,0.
001,vs.
RB1lightillumination(5wellseach,Mann–WhitneyUtest).
(b)BMSCs(53103cells)wereculturedingelatin-FA(15%)plusRB(0.
01or0.
1%).
The%viablecellsweremonitoredat24h,72handonday7afterlightilluminationwithvisiblelightfor2min.
Opencircle;RBonly,closedcircle;RB1gelatin-FA,opensquare;RB1lightillumination,closedsquare;RB1gelatin-FA1lightillumination(5welleach).
(c)Gelatin-FA(15%)with0.
05%RBwassubmergedinPBS,andexposedtovisiblelightfor2min,afterwhichthehydrogelswereobservedfor21daysat37uC.
Representativephotographs(5wellseach)areshown.
(d)BMSCs(53103cells)weresuspendedingelatin-FA(15%)with0.
05%RB,submergedinPBS,andexposedtovisiblelightfor2min,afterwhichthehydrogelswereobservedfor7daysat37uC.
Thepercentviablecellsinthehydrogelswereexaminedatindicatedtime-pointsafterthelightillumination(5wellseach).
(e)BMSCs(53104cells)weresuspendedingelatin-FA(15%)and0.
05%RB,submergedinPBS,andgelledbyilluminationwithvisiblelightfor2min.
DNAandacidicmucopolysaccharidecontentsinthegelatin-FAhydrogelsweremeasuredatindicatedtime-points.
Theresultantacidicmucopolysaccharide-to-DNAratiowasthencalculated(5wellseach).
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1038/srep044574withCBD-BMP4-loadedgelatin-FAwasstronglystainedbysafraninO,suggestingthepresenceofsufficientproteoglycansintherepairedtissue(Fig.
6c).
GrossandhistologicalscoresshowedthatCBD-BMP4ledtomorecartilagerepairthandidBMP4.
Tissuerepairbygelatin-FAitselfdemonstratedsomeimprovementcomparedwiththeuntreatedgroupat12weeks(Fig.
6b,d).
Toinvestigatethemolecularmechanismsofrepair,theregeneratedtissueswerehar-vestedat4weeksaftersurgeryandthemRNAexpressionofseveralchondrogenicfactorswereexamined.
SOX9,aggrecan,col1a1,andcol2a1wereallexpressedathigherlevelsintheCBD-BMP4-gelatin-FAgroupthanintheothergroups,includingtheBMP4group(Fig.
6e).
Thus,thesuperiorcartilagerepairintheCBD-BMP4groupFigure4|Gelatin-FAasacellscaffoldinanosteochondraldefectmodel.
BMSCs(1.
53105cells)weresuspendedingelatinsolution(15%gelation-FA10.
05%RB),implantedintoosteochondraldefects,andexposedtovisiblelightfor2min.
(a)Schematicillustrationoftheoperativeprocedure.
(b)Grossappearanceaftertheprocedure.
Representativephotographs(sixfemurseach).
(c)Grossgradingscoresat4and12weeksafterthesurgery.
*P,0.
05,**P,0.
01,***P,0.
001(sixfemurseach).
(d)RepresentativesafraninOstainingphotosat4and12weeksareshown(sixfemurseach).
Thescalebarindicates500mm.
(e)Representativemacrophagestainingphotosat4weeksareshown(sixfemurseach).
Thescalebarindicates200mm.
(f)Histologicalgradingscoresat4and12weeks.
*P,0.
05,**P,0.
01,***P,0.
001(sixfemurseach).
(g,h)RepresentativetypeIIcollagenimmunostaining(g)andaggrecanstaining(h)at12weeksafterthesurgeryareshown(sixfemurseach).
Thescalebarindicates500mm(g)and200mm(h).
Table1|EvaluationofsafraninOstainingSafraninOstain%SafraninOindefectdefect0.
760.
20.
860.
3Gelatin-FA1.
460.
2*1.
460.
2Gelatin-FA1BMSCs2.
260.
1*,**2.
660.
2**,***SafrainOstainwasevaluatedbyagradingscheme31.
*P,0.
05,**P,0.
001vs.
defect.
***P,0.
01vs.
gelatin-FA.
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1038/srep044575wasassociatedwithincreasedexpressionsofchondrogenicfactors.
TheBMP4groupfailedtoincreasetheexpressionofthesefactorsat4weeksafterthesurgery,possiblyowingtoitslossfromthehydrogel.
Furthermore,CBD-BMP4-loadedgelatin-FApromptedsubchon-dralbonerepair(Fig.
7a).
TheBV/TVvaluewassignificantlyhigherafterCBD-BMP4-gelatin-FAtreatmentthanwiththeothergroupsat12weeksaftersurgery(Fig.
7b).
Therefore,thismodifiedgelatin-FAwasalsousefulasascaffoldforgrowthfactor-basedtissueengineering.
DiscussionScaffoldsideallyneedtobebiocompatible,biodegradable,durable,andcapableofbeingformedintodesiredshapes.
Thenovelmodi-fiedgelatin-FAdevelopedinthepresentstudyappearstobeasuitablebiomaterialscaffold.
Comparedwithourpreviouslyreportedgelatin-FI,gelatin-FAismorerapidlycross-linkedinthepresenceofRBbyvisible-lightillumination.
Sucharapidconsol-idationissuitableforclinicaluse.
Inaddition,gelatin-FAhashigherelasticityandflexibilitythangelatin-FI,withanequivalentviscosity.
Thesesuperiorpropertiespromptedustoexaminethepotentialofgelatin-FAforuseinscaffold-basedtissueengineering.
Bycombin-inggelatin-FAwithBMSCsandCBD-BMP4,wedemonstratedthatphoto-cross-linkablegelatin-FAisapromisingscaffoldforosteo-chondralrepairbecauseitpromotesproductionofareparativematrix.
ClinicaltrialshavereportedthesafetyandtherapeuticeffectsofBMSCsadministrationinpatientswithOA19.
TheclinicaloutcomesaftertreatmentwithautologousBMSCswereasgoodasorbetterthanafterautologouschondrocyteimplantation20.
Chondrocytedif-ferentiationfromBMSCsinvitrousinggrowthfactorsrequiressig-nificanttimeandexpense,andsubsequentengineeredchondrocytesdonotfullymaintaintheirchondrogenicpropertiesafterinvivotransplantation21.
Therefore,employingBMSCsisareasonableapproachforthetreatmentofosteochondralinjuries.
OnekeypointwhenusingBMSCsforosteochondralrepairistouseanefficientdeliverysystemtolocalizethecellswithinalesionwithoutinhibitingtheinfluxofrepaircellsfromthesurroundingtissues.
BMSCsaremixturesofautologouslyacquiredpluripotentcellswithmultilineagedifferentiationandhighproliferationpotentials,allowingfortheirdifferentiationintoosteogenicandchondrogeniclineages22.
Studieshavedemonstratedthatthesevereanaerobicenvironmentinthejointisefficientforthephenotypicexpressionofchondrocytes23,24.
Maintaininghydrationandtheanaerobicenvironmentingelatin-FAhydrogelsmaystimulatethechondrogenicdifferentiationofBMSCs.
Here,weshowedthattransplantedBMSCspreservetheirchon-drogenicdifferentiationcapabilityinourgelatin-FAscaffold,asdeterminedbytheacidicmucopolysaccharide-to-DNAratio.
Alter-natively,itislikelythatBMSCsalsoattracthost-derivedmesenchy-malprogenitorcellsfromthesubchondralmarrowortheadjacentsynoviumviathereleaseofvariouscytokines19,whichinturnmayfacilitateosteochondraltissuerepair.
Wedemonstratedthatregen-eratedtissuebygelatin-FAplusBMSCstreatmentcontainedmoremacrophagesrelativetothatbygelatin-FAalone.
Furtherstudiesarenecessarytoaddresstherelativecontributionsofthesemechanisms.
Theuseofgrowthfactorswithtissueengineeringtechniquesisanotherpromisingtherapeuticstrategy.
Toincreasethetherapeuticpotencyofgelatin-FA,weemployedCBD-BMP4asagrowthfactor.
CBD-BMP4isanovelfusionproteinthatisretainedlongerthannativeBMP4intissuesconsistingofcollagen,andexhibitsanexquis-iteabilitytopromoteinvivoosteogenesis18.
BMP4iscapableofinducingosteogenic,aswellaschondrogenic,differentiation25,26.
BecauseCBD-BMP4isretainedlongeringelatin-FAhydrogels,weexpectedittofostertissuerepair.
Althoughbothgelatin-FAaloneandBMP4-gelatin-FAenhancedtissuerepairtosomedegree,CBD-BMP4-gelatin-FAproducedamarkedlyimprovedosteochondralrepair.
Duringthisprocess,gelatin-FAcoveringthedefectmayenhancetheintegrationbetweentheregeneratedtissueandthesur-roundingtissue,supportingtissuerepair.
TheacceleratedtissuerepairbyCBD-BMP4-gelatin-FAappearstobebasedonendogenouschondrogenicfactorsexpressedbyinfiltratingcellsfromthesur-roundingtissues.
SOX9isakeytranscriptionfactorforchondrogen-esis27.
SOX9activatesgenesexpressedinproliferatingchondrocytesthatincludesaggrecan28,oneofthemajorstructuralcomponentsofarticularcartilage.
Col1a1andcol2a1encodeforthemajorcom-ponentsofcollagentypesIandII,respectively.
TheincreasedexpressionofthesegenesintheCBD-BMP4groupsuggestedthatCBD-BMP4tetheringinthehydrogelscontributedtotheimprovedtissuerepair.
Figure5|Micro-computedtomography(CT)analysisat12weeksafterBMSCsimplantation.
(a)Representativethree-dimensional-CTimagesfromeachgroup(sixfemurseach)areshown.
(b)Bonegrowthwasassessedbybonevolume(BV)pertissuevolume(TV).
*P,0.
05,**P,0.
01(sixfemurseach).
Table2|ReleaseofBMP4andCBD-BMP4fromgelatin-FAhydrogelsday1day3day5day7BMP41.
2360.
101.
0360.
050.
9960.
070.
9660.
06CBD-BMP40.
2660.
02*0.
2360.
01*0.
2360.
00*0.
2160.
01*CulturesupernatantsfromBMP4orCBD-BMP4containinggelatin-FAhydrogelsweremeasuredforBMP4.
DataareODat490nm.
*P,0.
05vs.
BMP4(4welleach,Mann–WhitneyUtest).
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1038/srep044576Figure6|Gelatin-FAasagrowth-factorscaffoldinanosteochondraldefectmodel.
BMP4orCBD-BMP4(each250nM)wasmixedinto15%gelatin-FAcontaining0.
05%RB,andthemixtureswereimplantedintoosteochondralbonedefectsandexposedtovisiblelightfor2min.
(a)Representativephotographsofthegrossappearanceaftertheprocedure(fivefemurseach).
(b)Grossgradingscoresat4and12weeksaftertheprocedure(fivefemurseach).
(c)RepresentativeimagesofsafraninOstaining(fivefemurseach).
Thescalebarindicates500mm.
(d)Histologicalgradingscoresat4and12weeks(fivefemurseach).
(e)Theexpressionlevelsofchondrogenicfactorsat4weeks(5femurseach).
*P,0.
05,**P,0.
01,***P,0.
001(fivefemurseach).
****P,0.
05,vs.
untreatedcontrol(fivefemurseach,Mann–WhitneyUTest).
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1038/srep044577Thereareotherseveralconcernsthatwerenotaddressedinthisstudy.
Althoughwesucceededinproducinghyaline-likedifferen-tiatedtissuethatintegratedwiththesurroundingnativecartilageat12weeksafterthesurgeryunderfullweightbearingwithoutanyjointimmobilization,thephysiologicalandmechanicalpropertiesoftherepairedtissueremaintobeinvestigated.
Longer-termobser-vationisalsonecessary.
ItappearsthatthetransplantedBMSCsnotonlyfunctionasrepaircells,butalsostimulatethesurroundingtissuestowardsrepair.
However,theoriginofthecellspresentattherepairsite,whethertheyaretheoriginallytransplantedBMSCsorhostcellsthathavemigratedtothedefect,remainsunknown.
BMSCsmayescapefromgelatin-FAhydrogelsandmoveintothesurround-ingtissues.
AlthoughclinicaltrialshavereportedthesafetyofBMSCs19,themulti-potentpropertiesmaybepotentialthreatsforteratoma/tumorgenesisforalongterm.
Howgelatin-FAhydrogelssupporttissuerepairandwhengelatin-FAhydrogelsareabsorbedandreplacedbyregeneratedtissueisalsounclear.
Weshowedthatmacrophageswerepresentintheregeneratedtissueaftergelatin-FAtreatment.
Macrophagesareessentialtowouldhealingbytheirinter-actionswithothercellularpopulationsandreleasinggrowthfac-tors29,suggestingthatmacrophagesinthegelatin-FAscaffoldfacilitatethetissuerepair.
Aninterestingquestioniswhetheracom-binationstrategywithBMSCsandCBD-BMP4couldincreasetheregenerativepotentialofthescaffold,generatinganadditiveorsyn-ergisticeffect.
Furtherstudiesarenecessarytoaddressthesepossibilities.
Inconclusion,wehavedevelopedphoto-cross-linkablefurfury-lamine-conjugatedgelatin.
Withthismodifiedgelatin-FA,tissue-engineeringapproachesthatdeliverBMSCsandCBD-BMP4havebeenevaluatedinarabbitosteochondraldefectmodel.
Theresultsinthisstudydemonstratedthatthisnovelgelatin-FAisusefulforcell-based,aswellasgrowthfactor-based,scaffolds.
Gelatinisabiocompatible,biodegradableandmaneuverablematerialwithnoharmfuleffects.
Treatmentstrategiesusingphoto-cross-linkablegelatin-FAcouldbebeneficialforthetreatmentofosteochondralinjuries.
MethodsMaterials.
Porcineskingelatin(G2500),furfurylamine(FA),furfurylisocyanate(FI),deuteriumoxide(D2O),andRoseBengal(RB)werepurchasedfromSigma-Aldrich(St.
Louis,MO,USA).
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride(EDC)and2-morpholinoethanesulfonicacidmonohydrate(MES)werepurchasedfromDojindoMolecularTechnologies,Inc.
(Kumamoto,Japan).
N-hydroxysuccinimide(NHS)waspurchasedfromWakoPureChemicalIndustries(Osaka,Japan).
RecombinantmouseBMP4,affinitypurifiedanti-mouseBMP4polyclonalIgG,andbiotinylatedanti-mouseBMP4IgGwerepurchasedfromR&DSystems(Minneapolis,MN,USA).
CBD-BMP4fusionproteinwaspreparedasdescribedpreviously13.
Synthesisoffurfurylconjugatedgelatin.
Gelatin(5.
0g)wasdissolvedin250mLMESbuffer(50mM,pH5.
0)at40uC.
Subsequently,1.
533g(8mmol)EDC,0.
806g(7mmol)NHS,and2.
68g(28mmol)FAwereadded,andthesolutionwasmaintainedat40uCfor24h.
Thesolutionwasthendialyzedusingadialysismembrane(molecularweightcutoff:3,500Da)for2daysat40uC.
TheFA-conjugatedgelatinwasobtainedbylyophilizationandreferredtoasgelatin-FA.
Gelatincoupledwithfurfurylisocyanate(gelatin-FI)wasprepared,aspreviouslyreported13.
Thestructureofmodifiedgelatinwascharacterizedusingnuclearmagneticresonancespectroscopy(NMR).
ThesamplesweredissolvedinD2O,andthemeasurementperformedusingaJNM-AL300spectrometer(JASCOInternationalCo.
,Tokyo,Japan).
Visiblelightinducedcross-linkingandformationofhydrogels.
Anaqueoussolutionofgelatin-FAwasmixedwithanequalvolumeofRB.
Afterilluminatingfor2minwithvisiblelightfromalamp(LuminarAceLA-HDF158A,HayashiWatch-worksCo.
,Tokyo,Japan),themixturewastransformedfromasolutiontoahydrogelthroughphoto-oxidationcross-linking,asillustratedinFigure1A.
RheologicalexperimentswerecarriedoutwithanARES-G2rheometer(TAinstruments,NewCastle,DE,USA)usingtheparallelplates(25-mmdiameter,0u)configurationat37uCintheoscillatorymode.
Theevolutionofthestorage(G9)andloss(G0)moduliwererecordedasafunctionoftimeatafrequencyof1Hzandastrainof1%.
Animals.
FemaleNewZealandwhiterabbitsweighing2.
0–2.
5kgwereobtainedfromShimizuLaboratorySupplies(Kyoto,Japan).
Animalswerehousedinatemperature-controlledenvironmentwitha12-hlight/12-hdarkcycleandallowedfreeaccesstowaterandfood.
TheanimalcareandusecommitteeatOkayamaUniversityapprovedallanimalexperimentsconductedinthisstudy.
Allmethodswerecarriedoutinaccordancewiththeapprovedguidelines.
Bonemarrowstromalcells(BMSCs).
BMSCswerepurifiedasdescribedpreviously30.
Inbrief,bonemarrowcellswereaspiratedfromthehumerus,femur,andtibiaof5rabbits.
Thecellswerecentrifugedandwashedtwicewithphosphate-bufferedsaline(PBS,pH7.
4).
Thecellsweresuspendedinlow-glucoseDulbecco'sModifiedEagleMedium(DMEM)supplementedwith10%fetalbovineserum(FBS)andantibioticsandculturedovernightin100mm320mmdishesat37uCinahumidifiedincubatorwith5%CO2.
Unattachedcellswereremovedandthemediumwasreplacedevery3daysuntilthecellsreached80%confluence.
Coloniesformedcellsweredetachedwith0.
25%trypsin-EDTAsolution,washedtwicewithmedium,anddividedintotwodishes.
CellsatpassagestwotothreewereusedasBMSCs.
Underappropriatecultureconditions,differentiationassayswereperformedtoconfirmcelldifferentiationintochondrogenicandosteogeniclineages(notshown).
Thecellswereroutinelyalivemorethan97%bytrypanblueexclusion.
Cytotoxicityandphototoxicity.
RBtoxicityandphototoxiceffectswereexaminedusingBMSCs.
BMSCs(53103cells)wereincubatedina96-wellplateat37uCinaCO2incubatorfor24h.
Gelatin-FA(15%)andRB(0.
01,0.
1,and1%)wereaddedtotheDMEMwith1%FBSandgelledbyilluminationwithvisiblelightfor2minandculturedfor24h.
Inothersetofexperiments,Gelatin-FA(15%)wasgelledwithRB(0.
01and0.
1%)andexposedtovisiblelightfor2min,andculturedfor24,72hand7days.
EachwellwaswashedthreetimeswithPBSandmediumwasadded.
ThecellproliferationreagentWST-1(RocheDiagnostics,Indianapolis,IN)wasusedtodeterminethenumberofviablecellsaccordingtothemanufacturer'sinstructions.
ThecellnumbersinwellswithoutRBweredefinedasthecontrolsforeachgroup,andthenumberofcellsineachwellwasnormalizedtoitscontroltogive%viability.
Cellevaluationinthegelatin-hydrogel.
BMSCs(53104cells)weresuspendedinculturemedium(DMEMand10%FBS)containing15%gelatin-FAand0.
05%RB.
Themixturewascross-linkedbyvisiblelightilluminationfor2min.
Thehydrogelswereincubatedinaculturemedium,andthemediumwasreplacedevery3days.
Ondays1,7,14,and21,thehydrogelswerewashedtwicewithPBSandsonicatedin100mLextractionbuffer.
TheDNAcontentandacidicmucopolysaccharidecontentinthehydrogelsweredeterminedusingDNAQuantitykits(PrimarycellCo.
,Figure7|Micro-computedtomography(CT)analysisat12weeksafterBMP4orCBD-BMP4implantation.
(a)Representativethree-dimensional-CTimagesfromeachgroup(fivefemurseach).
(b)Bonegrowthwasassessedbybonevolume(BV)pertissuevolume(TV).
*P,0.
05,**P,0.
01,***P,0.
001(fivefemurseach).
www.
nature.
com/scientificreportsSCIENTIFICREPORTS|4:4457|DOI:10.
1038/srep044578Hokkaido,Japan)andacidicmucopolysaccharideassaykits(PrimarycellCo.
),accordingtothemanufacturers'instructions.
Osteochondraldefectmodel.
Rabbitswereanesthetizedwithketamine(1mg/kg)andisoflurane(0.
25–5L/min).
Cylindricalosteochondraldefectsweremadebilaterallyatthecenterofthemedialfemoralcondylewitha3.
0-mmdiameterdrilltoadepthof3.
0mm.
Fortymicrolitersofgelatinsolution(15%gelatin-FAand0.
05%RB)withorwithoutBMSCsorgrowthfactorswasappliedtothedefectsandcross-linkedwithvisiblelightfor2min.
BMP4andCBD-BMP4wereemployedasgrowthfactors.
Thedefectsuntreatedwereusedascontrols.
TherabbitsemployedinBMSCstransplantationmodelwere18rabbits(36femurs:defectcontrol;6femurs32timepoints(4weeksand12weeks),gelatin-FA;6femurs32,gelatin-FA1BMSCs;6femurs32).
Forgrowthfactortransplantation,20rabbitswereused(40femurs:defectcontrol;5femurs32timepoints(4weeksand12weeks),gelatin-FA;5femurs32,gelatin-FA1BMP4;5femurs32,gelatin-FA1CBD-BMP4;5femurs32).
Thefemursweretreatedatrandom.
Thejointcapsuleandtheskinwereclosedinlayerswith5-0nylonsutures.
Therabbitswereallowedfullweightbearingwithoutanyjointimmobilization.
Rabbitsweresacrificedat4and12weeksafterthesurgery,andthelesionwasassessedbyagrossgradingscheme31.
Inbrief,thedefectlesionwasscoredbasedonfourcategories,eachonascaleof0–4(bestscore:16):coverage,neocartilagecolor,defectmargins,andsurface.
Thefemoralcondylewasthenresectedandscannedbymicrocomputedtomography(micro-CT;HitachiAloka,Tokyo,Japan)at48-mmslices.
Regionsofinterestwerecenteredonthecylindricaldefectareaandanalyzedbyimage-analyzingsoftwareAZEVirtualPlace(AZE,Ltd,Tokyo,Japan).
Thebonegrowthwasmeasuredasbonevolume(BV)pertissuevolume(TV).
Subsequently,thefemoralcondylewasfixedin4%paraformaldehyde,decalcifiedin10%EDTA,embeddedinparaffin,andthesectionswerestainedwithhematoxylinandeosinandsafraninO.
Thesectionswereassessedbyahistologicalgradingscheme31,inwhichthedefectlesionwasscoredonascaleofmatrix(0–4),celldistribution(0–3),surface(0–4),safraninOstain(0–4),andpercentsafraninOindefect(0–4),with19beingthebestscore.
Forimmunohistochemistry,thesectionswereincubatedwithamonoclonalantibodytomacrophages(DAKO,Glostrup,Denmark),collagentypeII(ThermoFisherScientific,Waltham,MA),aggrecan(Abcam,Cambridge,UK)orcontrolIgG(5mg/mL)for1hatroomtemperature.
Thespecimenswererinsedandthenincubatedfor30minwithperoxidase-labeledpolymer(DAKO)atroomtemperature.
Diaminobenzidine(DAKO)wasusedasachromogenandcounterstainingwasperformedwithhematoxylin.
Quantitativereal-timepolymerasechainreaction(PCR).
RegeneratedtissueswerehomogenizedwithISOGEN(NipponGeneCo.
,Toyama,Japan),andtotalRNAwasisolatedaccordingtothemanufacturer'sinstructions.
First-strandcDNAwassynthesizedfrom2mgoftotalRNAwitholigo(dT)12–18asprimers,andthecDNAswereusedasatemplateforPCR.
Quantitativereal-timePCRwasperformedwithBrilliantIIIUltra-FastSYBRGreenQPCRMasterMix(AgilentTechnologies,SantaClara,CA,USA)andspecificprimers.
TovalidatetheSYBRGreenPCRproducts,adissociationstepwasperformedtoverifytheTm(annealingtemperature)oftheSYBRGreenPCRproductafterthePCRwasrun.
TheexpressionlevelsofeachmRNAwerenormalizedtotheexpressionofthehousekeepinggenehypoxanthinephosphoribosyltransferase.
TheprimersusedinthisstudyarelistedinSupplementaryTableS1.
Statisticalanalysis.
StatisticalanalyseswereperformedusingANOVAsformultiplesamplesifnototherwisespecified.
Insomecases,Mann–WhitneyUtestswereused.
AP-valueof,0.
05wasconsideredstatisticallysignificant.
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AcknowledgmentsWethankDr.
TakayukiFurumatsu,Ms.
ReinaTanaka,Mr.
YasuharuArashima,andMr.
HaruyukiWatanabefortheirexcellenttechnicalassistance.
Thisworkwassupportedinpartby1)grantsfromMinistryofEducation,Culture,Sports,ScienceandTechnology,Japan,andtheJapanSocietyforthePromotionofScience,Grant-in-AidforScientificResearch(S)ofKAKENHI22220009and(C)ofKAKENHI25462334,andby2)theStrategicInternationalResearchCooperativeProgram,JapanScienceandTechnologyAgency(JST),AS232Z00465F.
AuthorcontributionsT.
M.
,A.
M.
,T.
O.
andY.
I.
,ledtheprojectandconductedthedataanalysis.
T.
M.
andA.
M.
,wrotethepaper.
T.
M.
,Y.
S.
,K.
Y.
,A.
Y.
,M.
K.
,Y.
Y.
,D.
Z.
,T.
K.
andM.
T.
,performedtheexperimentsandcollectedthedata.
AdditionalinformationSupplementaryinformationaccompaniesthispaperathttp://www.
nature.
com/scientificreportswww.
nature.
com/scientificreportsSCIENTIFICREPORTS|4:4457|DOI:10.
1038/srep044579Competingfinancialinterests:Theauthorsdeclarenocompetingfinancialinterests.
Howtocitethisarticle:Mazaki,T.
etal.
Anovel,visiblelight-induced,rapidlycross-linkablegelatinscaffoldforosteochondraltissueengineering.
Sci.
Rep.
4,4457;DOI:10.
1038/srep04457(2014).
ThisworkislicensedunderaCreativeCommonsAttribution-NonCommercial-ShareAlike3.
0Unportedlicense.
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