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Pleasecitethisarticleinpressas:E.Garreta,etal.,Mater.Today(2017),http://dx.doi.org/10.1016/j.mattod.2016.12.005

MaterialsTodayVolume00,Number00January2017 RESEARCH

Tissue

engineering

by

decellularization

and

3D

bioprinting

Elena

Garreta

1

,

Roger

Oria

2

,

Carolina

Tarantino

1

,

Mateu

Pla-Roca

3

,

Patricia

Prado

1

,

Francisco

Ferna´ndez-Avile´s

4

,

Josep

Maria

Campistol

5

,

Josep

Samitier

6,7,8

and

Nuria

Montserrat

1,8,

*

1

PluripotentStemCellsandActivationofEndogenousTissueProgramsforOrganRegeneration,InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac 15-21,08028Barcelona,Spain

2

InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac15-21,08028Barcelona,Spain;UniversityofBarcelona,Spain

3

NanotechnologyPlatform,InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac15-21,08028Barcelona,Spain

4

DepartmentofCardiology,HospitalGeneralUniversitarioGregorioMaran˜o´n,InstitutodeInvestigacio´nSanitariaGregorioMaran˜o´n,Universidad Complutense-FacultaddeMedicina,c/DrEsquerdo46,28007Madrid,Spain

5

HospitalClinic,UniversityofBarcelona,IDIBAPS,08036Barcelona,Spain

6NanobioengineeringGroup,InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac15-21,08028Barcelona,Spain 7DepartmentofEngineering:Electronics,UniversityofBarcelona,08028Barcelona,Spain

8NetworkingBiomedicalResearchCenterinBioengineering,BiomaterialsandNanomedicine(CIBER-BBN),Madrid,Spain

Discarded

human

donor

organs

have

been

shown

to

provide

decellularized

extracellular

matrix

(dECM)

scaffolds

suitable

for

organ

engineering.

The

quest

for

appropriate

cell

sources

to

satisfy

the

need

of

multiple

cells

types

in

order

to

fully

repopulate

human

organ-derived

dECM

scaffolds

has

opened

new

venues

for

the

use

of

human

pluripotent

stem

cells

(hPSCs)

for

recellularization.

In

addition,

three-dimensional

(3D)

bioprinting

techniques

are

advancing

towards

the

fabrication

of

biomimetic

cell-laden

biomaterial

constructs.

Here,

we

review

recent

progress

in

decellularization/recellularization

and

3D

bioprinting

technologies,

aiming

to

fabricate

autologous

tissue

grafts

and

organs

with

an

impact

in

regenerative

medicine.

Introduction

Regenerativemedicineholdsthepromisetoreplaceorregenerate humancells,tissueororgansinordertorestoreorestablishthe normalfunctionlostduetodiseaseordamage[1].Bythe combi-nationofnovelbiomaterialswithcells,oneoftheaimsof regen-erativemedicine is to create autologous tissue graftsfor future replacement therapies [2,3]. In the last three years, discarded human donororgans, such askidney [4,5], lung[6], heart[7], andliver[8],havebeenusedtoobtaindecellularizedextracellular matrix (dECM) scaffolds,proving their potential applicationin

tissue engineering. Despite the translational value of these

advances,wearestillfartogeneraterelevanttissuesforimmediate clinicalapplications.

Theuseoforgan-deriveddECMscaffoldsfor bioengineering

of human-scale patient-specific organs using hPSCs is

envi-sioned as a major platform for therapeutic applications

(reviewed in [9]). Interestingly, theconcept of organprinting

has lately takencenter stage due to recent three-dimensional

(3D)bioprinting advancements(reviewed in [10–13]). Current

3D bioprinting techniques have shown to simultaneously

de-positcombinations of differentcell typesencapsulatedwithin

biomimetichydrogelsviaalayer-by-layerprocess,leadingtothe

generation of 3D bioinspired tissue constructs (reviewed in

[10,12,14,15]) [16–20]. Such approach could offer new

venues when translating hPSCs-related technologies to a

high-throughput 3D setup (e.g., patient induced pluripotent

stem cells (iPSCs)-derived organoid screening platforms)

(reviewed in[21]).

RESEARC

H:

Review

*Correspondingauthor:.Montserrat,N.(nmontserrat@ibecbarcelona.eu)

1369-7021/ß 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).http://dx.doi.org/10.1016/

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Sofar,differentlaboratories haveshownthatitispossibleto build up tissue- and organ-like structures either by the use of

organ-derived dECM scaffolds(reviewed in [22–27]) or 3D

bio-printingtechniques(reviewedin[10,12,14,15])[16–20].However,

thebioengineeringofvascularizedhuman-scaleorgananalogues

with optimal functionalactivitystillrequires much effortfrom multidisciplinaryresearchgroupsbeforethiscanbecomeareality. Here,wereviewthelatestadvancementsintheapplicationof decellularization/recellularization technologyforthegeneration

of autologous tissue graftstaking advantage of hPSCs. Wealso

examine how 3D bioprinting technologies may benefit from

hPSCsderivativestofabricatehumanorgananalogues.

The

advent

of

decellularization

technology

Tissue-specific

extracellular

matrix

(ECM)

through

decellularization

of

tissues

and

organs

Pioneerfindings onthe production oftissue-specificECM were

firstreportedinthe1970sand1980s[28,29].Despitethesemajor steps,itwasnotuntil10yearslater thatBadylakand coworkers generatedintactacellularsmallintestinalsubmucosamatricesby

mechanically removing allmesenteric tissues whileleaving the

trilaminateconnectivetissuelayersintact[30].Indeed,such ma-tricesdemonstratedhealingcapacityinadogmodelforAchille’s tendonrepair[30].Soon,differentworksondecellularizationof othersimpletissuessuchasskin[31],vasculartissue[32],heart

valves [33] and bladder [34] showed promising results on the

generation of biological scaffolds for biomedical applications, representingarealisticalternativetotheuseofsynthetic bioma-terialscaffolds.Fromthatmoment,furtherworksbegantoapply decellularizationmethodologiesforthefabricationofdECMslices fromcomplexorganssuchasliver[35].

In2008,theseminalworkbyOttandcoworkers,who

success-fullygenerated wholeratacellular heartsby meansofperfusion decellularization,representedabreakthroughinthefieldoftissue

engineeringand thebeginningoftheeraofwholeorgan

decel-lularizationtechnology[36].Overthelastyears,differentresearch groupsfollowingsimilarapproacheshavereportedthepossibility toderivefull-scaledECMscaffoldsfromdifferentorgansincluding liver, heart, lungs, and kidneys;and multiple species including mouse,rat,pig,rhesusmonkey,andhuman(reviewedin[22,23])

[37]. The main milestones on decellularization technology are

summarizedinFig.1.

InvivothecompositionandultrastructureofECMisinconstant remodelingbytheresidentcellsdependingonthemetabolicand

mechanical demands of the tissue, a concept called ‘dynamic

reciprocity’[38].Suchdynamicremodelingmaybealteredduring injuryordisease,leadingtomodificationsinthecompositionand biophysicalpropertiesoftheECM,andultimately,compromising

organ function. Accordingly, it hasbeen suggested that ‘organ

specificity’,maydeterminewhycellsbelongingtoaspecificorgan exhibitaninnatepreferencetowardsdECMscaffoldsderivedfrom the organ oforigin[39,40]. Alongthis line, Nakayamaand

co-workersshowedthathumanembryonicstemcells(hESCs)

differ-entiated intorenal-likecells whenseeded onto rhesusmonkey

kidneydECM,butthiswasnotthecasewhenlungdECMwasused

instead[40].Themainhypothesissupportingsuchfindingsrelies ontheideathatspecificECMproteinsaredistinctivelydistributed alongthedifferentcompartmentsoftheorganasfootprintsfrom

resident cells. Interestingly, site-specific ECM micromechanical

properties have been described to delimitate different tissue

regions[41].

Overall,dECMscaffoldsprovideacomplexsite-specific

combi-nation of biochemical and mechanical cues, which have been

hypothesizedtoguidecelladhesion,proliferationand

differentia-tion during recellularization and further tissue formation

(reviewed in [42]) [39–41,43–45]. The main advantages using

dECMscaffoldsforthedenovobiofabricationoftissuesandorgans aresummarizedinBox1.

Methods

of

decellularization

Decellularizationtechniquesaim toremove allthe cellsfroma tissueororganwhilepreservingthenativeECMcompositionand architectureintegrity.Assuch,tissueandorgandecellularization, canleadtotheproductionof3DdECMscaffoldsretainingtheir biologicalactivityandmechanicalproperties.Ifeffective,dECM scaffoldsshouldnotelicit immune-mediatedrejectionafter

im-FIGURE1

Timelineofkeyeventsleadingtowholeorgandecellularization methodologiesandmajormilestonesusinghPSC-derivedcellsto repopulateorgan-deriveddECMscaffolds(*).

BOX1

PotentialofdECMscaffoldstorecapitulatetissuesandorgans

PreservationofdECMultrastructureandcompositioninducefavorable tissueorganizationandremodeling(reviewedin[23,24,36]).

dECMcanmodulatecellbehavior:attachment,migrationand differentiation(reviewedin[23,24,36])[39,40,43,44].

dECMdonotelicitimmune-mediatedrejection,sinceECMcomponents arelargelyandhighlyconservedacrossspecies[46,47].

dECMexhibitsuitablemechanicalperformance,similartothatoftheir nativecounterparts(reviewedin[23,24,36])[41].

dECMpromoteconstructiveremodelingresponse,asshownforavariety oftissuesinbothpre-clinicalandclinicalstudies(reviewedin[42])[45]. ThepotentialadvantageofdECMorganspecificityensuresthe maintenanceofselectedcellfunctionsandphenotypes(reviewedin[38])

[39].

2

RESEARCH:

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plantation [46,47]. Extensive research has shown the mode of actionand peculiaritiesofeach kindofdecellularizationagents employed,aswidelydiscussedelsewhere(reviewedin[48–50]).In general, the optimal decellularization protocol will generate a

DNA-freedECMscaffoldbyfindingtherightcompromisebetween

thedurationandcomplexityofthetreatmentandthe

conserva-tion of the desired compositional, structural and mechanical

propertiesofthegenerateddECMconstruct.

Withrespecttothetechniquesusedtoinfusethe decellulariza-tionagentswithinallregionsoftheorgan,perfusionthroughthe

vasculature and immersion/agitation are nowadays the most

employed, though others techniques have been also described

(e.g.,pressuregradient[51,52],supercriticalfluid[53]).Perfusion decellularizationtakesadvantageoftheinnatevasculatureofthe organ to deliver the decellularization agents across the entire

organ.Suchapproachisoftenperformedinorgansinwhichits

mainarterycanbecannulatedforperfusionwithdecellularization solutionsunderphysiologicperfusionpressures.Intheirseminal work,Ottandcoworkersdevelopedaprotocolforwholeratheart

decellularization using a homemade bioreactor to perfuse 1%

sodiumdodecylsulfate(SDS)throughthecoronaryvasculature,

showingthat12hoursperfusionata physiologicalpressure suf-ficedtoyieldafullydecellularizedratheart[36].Thesameresearch

group subsequentlyapplied a similar approach to decellularize

wholeratlungs[54]andkidneys[55].Forlungdecellularization,

boththeairwayandvascularcompartmentswereinvestigatedto

deliverthedecellularizationsolutions,renderinginallcases

acel-lular organ scaffolds with preserved ECM composition,

micro-structure and 3D architecture [54,56–59]. Overall, perfusion

decellularizationisthepreferredtechniquetodecellularizewhole organs,especiallyinlargeanimalsorhumans[4–8,58,60–63].

Onanotherhand,inimmersion-baseddecellularization proto-cols,thetissueororganofinterestissubmergedintothe decel-lularizationsolutionswhilebeingsubjectedtoagitation.Inthis way,decellularizationagentsenterthroughthetissuebydiffusion.

Thedurationoftheprotocolwillthendependontheinitialtissue thicknessandcelldensity.Suchapproachismainlyusedontissue samplesthatdonothaveeasyaccesstothevascularnetwork(e.g., skeletalmuscle,skin)[64–66],aswellasonorganslicestypically

obtained from a segmental resection. Box 2 summarizes the

advantagesanddisadvantagesofthedifferentmethodsof decel-lularizationdiscussedinthisreview.

Due to the large diversity of decellularizationprotocols and tissue sources reported so far, there has been the necessity to

establish common criteria to evaluate the effectiveness of any

decellularizationprocess.Decellularizationrequirementsand

as-sociatedmethodologiesaresummarizedinBox3.

Although successful decellularizationwas achieved formany

organs,stillmuch effortshouldbedirectedonthe definitionof standardized decellularization protocols with the final goal to advanceinthecreationofbiocompatibleandpersonalizedorgan scaffoldsforclinicalapplications.Forthat,issuesincluding

bio-degradation,cytocompatibility,pathogenicityand

immunogenic-ityshouldalsobefurtherstudied(reviewedin[67]).

Recellularization

of

whole

organ

dECM

scaffolds

Oneofthemajorissuesinthefieldoforganbioengineeringisthe precisepositioningofspecificcelltypesinsidetheircorresponding

specificorgancompartment.Methodsofcellseedingintowhole

organ-derived dECM scaffoldswilllargelydependonthe organ

itself and usually will require the use of bioreactors. Complex organsincludingkidney,heart,lung,andliverentailthe develop-mentofchallengingcellseedingandculturemethodstopromote

tissue formation and maturation. Oxygen diffusion across an

engineered tissue is limitedto a maximumtissue thickness of

200micrometers (reviewedin[68]),meaningthat highertissue thicknessesmustrequirefunctionalvasculaturetosupplythecells

with oxygen and nutrients as well as facilitate the removal of

metabolicwasteproducts.Inthisregard,bioreactortechnologyfor

whole organ engineering stillneeds to overcome many issues:

MaterialsTodayVolume00,Number00January2017 RESEARCH

MATTOD-849;NoofPages13

Pleasecitethisarticleinpressas:E.Garreta,etal.,Mater.Today(2017),http://dx.doi.org/10.1016/j.mattod.2016.12.005

BOX2

Methodsofdecellularization

Perfusion Immersion/agitation

Modeofaction Infusionofthedecellularizationagentsthroughtheorgan vasculature

Immersionofthetissueororganintothedecellularization solutionswhileshaking

Preferredwhendecellularizinglargeanimalorhuman organs

Usedwhentheaccesstothevasculatureisdifficultor absent

Advantages Facilitateshomogeneousexposuretothedecellularization reagentsandremovalofcellularcontent

Mechanicalagitationfacilitatescellularcontentremoval Thepossibilitytoapplyphysiologicalperfusionpressures

wouldfavorpreservationoftissueECMcompositionand architecture

Easyandfastprocedurewhendecellularizingsmallanimal organsortissues,andhumantissuesobtainedfrom segmentalresections

Controlledperfusionconditionsbytheuseofbioreactors enhancestherobustnessandefficiencyoftheprocess

Doesnotneedoverlyspecificbioreactorequipment

Disadvantages UnappropriateperfusionpressurescandisruptECMand impactontheviscoelasticbehaviorofthedECMscaffold. Optimizationisrequiredforeachtissue/organ

Decellularizationconditions(e.g.,agitation,reagents’ exposuretime)havetobeoptimizeddependingonthe tissuethickness.ExcessiveagitationcandisruptECM Needscannulationofthemainorganartery Itisanunreliablemethodwhendecellularizinglarge

animalorhumanwholeorgans Thismethodusuallyneedstheuseofspecificperfusion

bioreactors

Thismethodusuallyneedsincreasingtimesofexposureto thedecellularizationagentswhencomparedtoperfusion References [4–8,54,56–63] [39,64–66]

3

RESEARC

H:

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BOX3

Establishedcriteriaforeffectivedecellularization

Requirement Methodology Outcomeforeffectivedecellularization References Removalofcells

andDNAcontent

Decellularizedtissuesshouldaccomplish Avoidadversecellandhostresponseaswell asnegativetissueremodelingand inflammationresponsesafterimplantation

[34](reviewedin[48])

(i)<50ngofdouble-strandedDNA(dsDNA) permgofdryweightdECM,asquantifiedby commerciallyavailabledsDNAintercalators suchasPicoGreenandgelelectrophoresis (ii)<200bpDNAfragmentlength,analyzed bygelelectrophoresis

Histologicalstainings(Hematoxylinand Eosin,Masson’sTrichome,Movat’s Pentachrome,orSafrinO)or

immunohistochemistryanalysisindECM shoulddenotethelackofvisiblenuclei(as stainedDAPIorHoechst).Thesecanbeused toqualitativelydetectifnucleicontent, cytoplasmicproteinsorsomeextracellular componentsarestillpresentafter decellularization

Quantificationof residualdetergents

QuantificationofremnantSDScanbe determinedusingStains-Allreagent

Avoidtoxiceffectsformthese decellularizationagents,ensuringcell viabilityduringdECMscaffold recellularization.

[67]

SimilarlyresidualTritonX-100canbe quantifiedbyusingderivative spectrophotometry.

Undetectableamountsofsuchcomponents aredesired

Preservationof ECMcomponents

Qualitativeevaluationby

immunohistochemistryanalysisofthemain ECMproteinsincludingcollagens,laminin, fibronectinandelastin

Retentionofthemainbasementmembrane componentsandstructuralECMproteinsas comparedtotheirnativecounterparts

[8,43,56,84]

Quantitativecolorimetricassaysare employedtodeterminetheamountof collagen,elastinandglycosaminoglycans usingcommerciallyavailableSircol,Fastin andBlyscankits,respectively

Massspectrometry-basedproteomics allowspreciseidentificationofmatrisome proteinsandothertissue-specificproteins Maintenanceof

3Darchitecture andvascular integrity

Micro-andnano-architectureofrelevant parenchymalstructuresofeachorgancanbe assessedbyscanningelectronmicroscopy (SEM)

Conservationofthemainstructuralfeatures specificofeachorgan,whichwillfacilitate tissueorganizationandmaintenanceof specificcellphenotypes

[4,5](reviewedin[22,36])

Conservationofthehierarchicalvascular bedoftheorganafterdecellularizationcan beevaluatedbyMicroCT,dyeormicrobeads perfusionassays,angiographyorcorrosion casting

Conservationofthehierarchicalvascular bedoftheorgan,whichisessentialtofurther achieveaneffectiverecellularization outcome

Biomechanical performance

Traditionalmaterialscienceand

engineeringtechniquesincludinguni-or bi-axialmechanicaltestingandatomicforce microscopy(AFM)havebeenmainlyusedfor mechanicaltestingonproduced

decellularizedscaffolds

OptimaldECMscaffoldmechanical properties(mechanicalstrengthand viscoelasticbehavior),thatfollowing recellularizationshouldbesimilartothoseof theirnativecounterparts

[4,41,56,63]

Lungmechanicshasbeenassessedusing pressure-volumecurvesandmeasuringforce tensionrelationshipsinlinearstripsof decellularizedlungs

4

RESEARCH:

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fromthedefinitionofoptimalcellseedingmethodologiestothe possibilitytomonitorbiochemicalandbiophysicalmarkers indic-ativeoforganmaturationandfunctioninanon-invasivemanner. Otherissuesarerelatedtotheapplicationofphysiologicalrelevant stimulithatcanenhancetissueformationandfunctionality.The

principle bioreactor requirements for whole organ culture are

listedin Box4.Extensivediscussiononrecentbioreactor devel-opmentsisreportedelsewhere(reviewedin[69])[70–77].

Vascularandnon-vascularrouteshavebeenemployedto deliv-ercellsintowholeorgan-deriveddECMscaffolds,mostly

follow-ing dynamic seeding procedures, which consist of introducing

cellsataspecificconcentrationintothevascularperfusionline. Followingthismethodology, researchershaverecellularizedthe

vasculature and parenchyma of liver, heart, lung and kidney

(reviewed in [22–27]). Cells introduced into the vascular flow

maytraversethe vascularliningthroughporesproducedduring

decellularization, thenreaching the organ parenchyma. In this

regard,ithasbeen proposedthattheproperadjustmentofflow

rates may reduce shear stress on cells, while minimizing the

potential damage on the dECM scaffold. Moreover, multiple

inoculationsof cellsarepreferablethana uniqueone withthe sametotalcellnumber.Performingmultiplecellinfusionsdirectly intothelivervascularcircuitinastep-wisemannerledtomore than85%cellengraftment[77–79],alsoshowingamoreefficient distributionofcellsacrossallregionsoftheorgan-deriveddECM scaffold.Alternatively,directinjectionofcellswithasmallgauge

needle by performing multiple injections throughout different

areas of organ parenchyma has shown lesssuccess [80]. Other

non-vascularroutescommonlyusedtoreintroducecellsarethe

tracheainlungs[54,56–59]ortheureterinkidney[55,81–83]. Sofar,thelargebodyofworkregardingrecellularizationofwhole

organdECMscaffoldshasbeenperformedinsmallanimalmodels

usingdifferentbioreactor settings, celltypesand seeding condi-tions(reviewedin[22–27]).Themainproblemsencountered dur-ingrecellularizationweretheuncompletere-endothelizationofthe organvasculature,andtheinsufficientrepopulationoftheorgan parenchyma[55,56,60].Notably,recently thisyear,Guyetteand coworkershavepartiallyrepopulatedwholedecellularizedhuman

heartsusingacustomhumanheartbioreactorcapableofproviding coronaryperfusionandleftventriclewallmechanicalstimulation,

showing metabolically active repopulated myocardial segments

after14days oforgan culture[84].Also Nicholsand coworkers havereported,forthefirsttime,thedevelopmentofabioreactor systemtosupportrecellularizationofwholehumanpaediatriclung dECMscaffolds,identifyingthemainconditionsandcell require-mentsnecessaryforbioengineeringwholehumanlungs[85].

Moreover, many different cell types and sources have been

reported for recellularization strategies of different organs

(reviewedin[22–27]).Initialworksmadeuseofneonatalorfetal cellsderivedfromtheorganofinterest,showingretentionoftheir

tissue-specific phenotype after seeding into the organ-derived

dECMscaffoldtogetherwithrelevantorgan-specificfunctionality. Overall,theseworksservedasaproof-of-conceptofthefabrication

of whole organs de novo by decellularization/recellularization

techniques.However,thosefindingsalsohighlightedthe necessi-tytofindmoreamenablecellsourcesthatcouldbeeasily expand-edanddifferentiatedintofunctionalandmultiplecelllineages.In

this regard,hPSCshave beenproposed aspromising candidates

duetotheirself-renewalcapacityandthepotentialtogiveriseto anycelltypeinthebody[86–88].TheuseofhPSCsasacellsource

for the development of bioengineered organs based on dECM

scaffoldsisfurtherdiscussedinthefollowingsection.

Pluripotent

stem

cells:

a

long-standing

cell

source

for

regenerative

medicine

Pluripotent

stem

cells

Pluripotencyisdefinedastheabilityofasinglecelltodivideand

produce differentiated cells from the three germ layers of the

embryo[86–88].Theideatogeneratefunctionaltissuesandorgans frompluripotentstemcells(PSCs)hasbeenalong-standinggoalin stemcellbiology,representinganunprecedentedopportunityto

study development and evento heal degenerative diseases and

aging-related disorders.Inthis regard,thepossibilitytocapture

and culture indefinitely hESCs from the pluripotent inner cell

mass(ICM)oftheblastocysthasbeenamajorbreakthroughinthe areaofregenerativemedicine[86].

BeforehESCswerefirstderived,seminalstudiesalreadytriedto

answerhowshapeandpatternemergefromthesimplebeginnings

ofanembryo,andevenhowspecializedcellsdifferentiateduring

embryodevelopmentbecomingorganizedintoa3Darchitectural

context (reviewedin [89]) [90].Pursuingthe idea toreprogram differentiatedcellstoan‘embryonic’state,Takahashiand Yama-nakain2006discoveredthatthepluripotentstatefoundinhESCs derivedfromtheICMcouldbeartificiallyinducedinasomaticcell

through the overexpression of just four transcription factors

(OCT4, SOX2, cMYC,and KLF4-OSKM) [87](reviewed in [91]).

Theproducedcells,socallediPSCs,exhibitedallthemolecularand functionalfeaturesofESCs.Importantly,inthelastyearshuman

iPSCs(hiPSCs)haveshowntobecomeinstrumentalplatformsfor

thestudyofhumandevelopmentanddiseasewiththe identifica-tion,insomecases,ofmolecularandcellularmechanisms respon-siblefordiseasegestationandprogression(reviewedin[92]).

Overall, one major limitation in the field of hiPSC disease

modeling is the lack of a systemic context and disease-related

environmental cues [e.g., disorganized ECM, insufficient

bio-chemical signals from the niche, among others], opening new

MaterialsTodayVolume00,Number00January2017 RESEARCH

MATTOD-849;NoofPages13

Pleasecitethisarticleinpressas:E.Garreta,etal.,Mater.Today(2017),http://dx.doi.org/10.1016/j.mattod.2016.12.005

BOX4

BioreactorrequirementsforrecellulatizationofwholeorgandECM scaffolds

Includeindependentaccesslinesforcellseedingthroughvascularand non-vascularroutes

Integratepressuretransducerstocontrolflow/volume-basedpressure Allowcontinuousorpulsatileperfusionthroughthevasculatureto providenutrientswhileremovingmetabolicwasteproducts Monitoringandmaintenanceofphysiologicalvascularpressure Monitoringandmaintenanceofpropergasessupply

Monitoringorganmechanicsduringrepopulation

Canincludeorgan-specificbiophysicalstimuli(e.g.providemechanical ventilationinlung,providemechanicalstretchandelectricalstimulationin heart)

Allownon-invasiveorminimallyinvasivemonitoringofrelevant biochemicalandbiophysicalmarkersindicativeoforganmaturationand function

Allowautomationofcriticalparameters

Maintainsterility(disposableoreasilysterilizedcomponents)

Goodmanufacturingpractices(GMP)amenable/Clinicalgradebioreactors

5

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H:

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challengeswhenintegratingbiomaterialsmimickingdisease pa-thology.Inthisregard,ithasbeenrecentlyshownthattheproper fine-tuningof3Dscaffoldsrecapitulatedtheabnormal

contractili-tyinhiPSC-derivedcardiomyocytesfrompatients withlongQT

syndrometype3,andnotwhenhealthycounterpartswereused.

Suchplatformalsoallowedforthescreeningofcardiotoxic com-poundsindifferent3Dsettings,highlightingthepotential appli-cationoftheseapproachesforhumandiseasemodeling[93].

Differentiation

strategies:

from

pluripotent

colonies

to

organoids/organogenesis

in

a

dish

OneofthemajorareasofresearchinthefieldofhPSCshasbeen thedevelopmentofprotocolsforthegenerationoffunctionalcell typessuitablefordiseasemodelingandcellreplacementtherapies. Until recently,most protocolsof differentiationrelayedon the generationofsinglecellpopulationsratherthancompletetissues. However,inthelastthreeyearspivotalstudieshavedemonstrated

thatit ispossible to generate 3Dcultures ofdevelopingtissues namedorganoids(reviewedin[94,95]).

Afterthe firstderivation ofhESCs [86], differentlaboratories worldwideexploredhESCscapacitytoundergocontrolled differ-entiationeitherinmonolayer,byseedingcellsinthepresenceof differentECMproteincoatings(majormatricesusedforthe cul-tureofhPSCsaredescribedinBox5) (reviewedin[94–96])[97– 101],orasspheroid-likestructuresnamedembryoidbodies(EBs) (Fig. 2). Although EBs can recapitulate several aspects of early

development(reviewed in [94]), asanyother methodology, EB

formationstillhampers the translationofthis approach intoa clinicalsetting(e.g.,lowreproducibilityand scalability). Never-theless,alltheseadvanceshavebeenfundamentalfortheproper instructionofhPSCs to formself-organized tissue-specific orga-noidsincludingtheopticcup,brain,intestine,liverandkidney (reviewedin[94]).

Organoids are similar to in vitro derived EBs, but they can

recapitulatea largenumberofbiological processesrelated with spatialandtemporalorganizationofheterogeneoustissue-specific cellswithinthe3Dstructures(Fig.2).Eveninsomecases, orga-noidshaveprovedtoexhibitphysiologicalfunctionsbeingclose totheinvivosetting.Inthisregard,kidneyorganoidsderivedfrom

hiPSCs havebeen recentlyshown tocontainmultiplenephron

segments surrounded by nascent blood vessels, being able to

respondinfrontofnephrotoxiccompounds[102].Despitethese

findings,most hPSC-derived organoid modelsonly represented

single or partial components of a tissue, hindering the proper

control of cell-cell interactions, cell-matrix interplay, and cell

organization.Moreover,commonhurdlesinorganoid

technolo-giesarebeingrelatedtoincompletematurationofhPSC-derived celltypesandthelackofvascularization.

Bioengineering

approaches

for

hPSCs

differentiation:

dECMs

as

biomimetic

platforms

for

generating

tissues

on-demand

Bioengineeringapproachescanbeusedtoovercomemajorissues

associatedwithhPSCsdifferentiationasmaturationand function-ality.Inthisregard,matrigelhasbeena fundamentalmatrixfor organoidmethodologies,includingcerebral,opticcupand intesti-nalorganoidsfromhPSCs(reviewedin[94,95]).However,matrigel

BOX5

ExtracellularmatrixcomponentsusedinhPSCscultureanddifferentiation

ECMcomponents References

CollagensarethemostabundantECMmacromoleculesfoundinourbody.Amongthe28typesofcollagenthatexist,collagen IVandIhavebeenwidelyusedinhPSCsdifferentiationstudiesdemonstratingtobeimplicatedinmesodermaldifferentiation.In addition,collagen’sabilitytoself-aggregateandcrosslinkmakesitanattractivemacromoleculeforbiomaterialscience

[97]

Lamininisatrimericproteinfoundinthebasementmembrane,whichhasbeenlargelyinvestigatedforitsinfluenceinhPSCs differentiationtowardsectodermaltissues

[98]

FibronectinisanECMproteinhighlyexpressedduringtheearlystagesofembryonicdevelopment,beingessentialforproper developmentofthemesodermandtheneuraltube.Generally,itiswidelyusedascelladhesionproteinduetothepresenceof thepeptidesequencearginine-serine-asparticacid(RGD)initsstructure,whichisimplicatedinintegrin-mediatedcelladhesion

[99,100]

MatrigelisgeneratedfromthebasementmembraneofmousesarcomacellstherebycontainingavarietyofECMmolecules andgrowthfactors.LamininisthemajorconstituentalthoughcollagenIVandproteoglycansalsotakepartofitscomposition. Sinceitisderivedformbasementmembraneitprovidesarichenvironmentthathasbeenlargelyusedforthemaintenanceof hPSCsaswellasdifferentiationtomanylineagesincludingcellsfromthethreegermlayers,mesoderm,endodermandectoderm

Reviewedin[94–96]

Cell-depositedECMcanbeextractedfromdifferentcelltypesbyfirstallowingthecellstoproducetheirownECMandthen removingthemwithoutdisturbingthenewlysynthesizedECMstructureandbiochemicalcomposition.Suchcell-depositedECM hasbeenthenusedasacoatingforstemcellstemnessanddifferentiationstudies

[101]

FIGURE2

ThemainstrategiesusedforhPSCsdifferentiationincludeguided differentiationin2Dmonolayercultures,theformationofembryoidbodies andtheinductionof3Dself-organizationgivingrisetoorganoids.The generationoforganoidsneedsahighercellculturecomplexitythanthe othertwoapproaches.

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Materials Toda y  V olume 00, Number 00  Januar y 2017 RESEARCH MA TTOD-849; No of Pages 13 Please cite this article in press as: E. Garreta, et al., Mater . T oday (2017), http://dx.do i.org/10.1016/j.mattod.2016.12.0 05 BOX 6 3D bioprinting techniques Bioprinting techniques

Additive unit Actuation method Cell

viability

Commercial bioprinter

Bioink viscosity

Disadvantages Advantages References

Inkjet printing Drop  Piezoelectric pulse

 Thermal induced pulse

>85% Yes Low  Microvalves for inkjet are

fragile

 Requires fast material gelation/stabilization  Height of 3D constructs limited due to low bioink viscosity

 High control on the amount of material deposited (1 pL to 0.1 nL per drop) Reviewed in[12] Laser assisted bioprinting

Drop  Laser induced pulse >95% No Medium-high  High cost of future

commercial bioprinters  Long fabrication time  Long preparation time of material ribbons

 Low diversity of bioink for ribbon preparation

 High printing resolution  Single cell deposition

Reviewed

in[12]

Micro extrusion Material strain  Pneumatic pressure 40–95% Yes Medium-high Pneumatic pressure

 Cells may suffer important shear stress

 Medium printing accuracy  Low printing resolution

Pneumatic pressure  Abrasive materials can be used

 Disposable cartridge that avoids cross-contamination  Non mechanical parts  Used by most of commercial bioprinters due to its robustness and simplicity  Allows printing cell high density

Reviewed in[10,12]

 Mechanical pressure Mechanical pressure

 Cells may suffer important shear stress

 Medium printing accuracy  Low printing resolution  Mechanical parts make this system more fragile  Screw based system require cleaning of mechanical parts

Mechanical pressure  Abrasive materials can be used

 Piston based systems uses disposable syringes  Allows printing cell high density

Stereolithography (SLA)

Cured bioink voxel

 Laser based curing  UV and visible light projection curing

>85% Yes Medium  Only can be used with light

crosslinkable bioinks  Multicellular structures are challenging

 Printer are not specifically designed for biofabrication

 Affordable

 High printing velocity

Reviewed in

[10,123,127,128]

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compositionisnotwelldefinedandbatch-to-batchdifferencesmay leadtoimportantdifferencesinexperimentaloutcomes[103].

Alongthisline,biomaterialscanbeusedtocreatestem-cell-like nichesprovidingkeyelementstocontroltheregulationofstem cellfateandfunction.Indeed,materialpropertieshavebeenoften designedtomimicphysiologicallyrelevantECMstiffness, topog-raphy,andadhesion-ligandtype,densityandaffinity.These

fea-tures,whencombined withhPSCshaveledtothe derivationof

protocols for hPSCs differentiation building personalized tissue

constructs using human organ-derived dECM scaffolds

[43,84,104,105],andeven forpartiallyor totally reconstructing

mouse[106]andhumanwholeorgans[84,105].

Ottandcoworkersrecentlyreportedforthefirsttimethewhole repopulationofdecellularizedhumanheartswithhiPSCs-derived cardiomyocytes[84].Inthesameline,ourgroupdevelopedarapid protocolforthegenerationofhumanheartgraftsbyco-culturing

hPSC-derived cardiomyocytes on top of 400micrometers-thick

slicesofhumanventriculardECMscaffolds[43].

Importantly,rat and humanlung dECM scaffoldshave been

alsorecentlyshowntoberepopulatedwithendothelialand peri-vascular cellsdifferentiated fromhiPSCs [105].Concerning kid-ney,onlytwoworkshaveinvestigatedtheroleofrhesusmonkey kidneydECMscaffoldsonhPSCsrenaldifferentiation[40,107].

Alternatively, 3D bioprinting technology have opened new

venuesforthebottom-upgenerationoftissueandorgananalogues bythedepositioninanadditivelayer-to-layerapproachof differ-entiatedhPSCs and biomaterials,specifically arrangedto

repro-duce native 3D architectures (reviewed in [11,12,95]).

Nevertheless,attemptstogeneratehPSCsderivedbioprinted con-structsarestillinitsinfancy,withonlyonereportonthe genera-tionofminiliversfromhPSCs[16].

Tissue

engineering

of

human

organ

analogues

by

3D

bioprinting

3D

bioprinting

techniques

Bioprintingtechniquesaimtoperformsimultaneousdepositionof

single or multiple combinations of living cells together with

supportivematricescontainingbiochemicalandbiophysicalcues (altogether termed asbioink). In this manner, organs or tissue analoguesareconstructedfollowingapredefinedarchitecturein 3D(reviewedin[12–14]).Thesemethodologiesaregenerally clas-sifiedbasedonthetechnologyusedtogeneratethestructuresof thecell-ladenmaterialasfollows:

Inkjet bioprinting, also referred as ‘drop-on-demand printers’ appeared early in 2003 [108]. Firstly developed inkjet printers

modifiedcommerciallyavailabletwo-dimensional(2D)ink-based

printers by replacing the ink in the cartridge by a biological

material, and the paper, by an electric-controlled elevator that

moves on the z direction providing three-dimensionality

(reviewed in[12])[109].Nowadays,inkjet printersmake useof nozzles thatgenerate isolateddroplets ofcell-ladenmaterialby meansofpiezoelectric[110]orthermal(reviewedin[111]) actu-ationsystems.Inthismanner,bymeansofeitheracousticwavesor thermalforces,respectively,liquiddropsareejectedontoa sub-strate. In the last years, inkjet bioprinting has allowed for the efficientintroductionofgradientsofcellsorgrowthfactorsalong the3Dconstructsbythemodificationofdropdensitiesandsize

[17,112,113].Despitethismajoradvances,onecommondrawback

ininkjetprintingistheneedtoworkwithbiologicalmaterialsin liquidforms,whichinmostofthecasesdemandafast polymeri-zation procedure post-printing (e.g., by eitherchemical, pHor ultravioletmechanisms,amongothers).Alltheseprocedures di-rectlyaffectthebioprintingprocess,compromisingthechemical andmechanicalpropertiesofECM-derivedmaterials.Asreviewed elsewhere,otherdisadvantagesarerelatedtotheimpedimentof building3Dcell-ladenconstructswithgeneraltissuesize,nozzle cloggingwhenusingsolutionswithhighcell densities,andcell viabilityconstraints(relatedtotheuseofcross-linkers)(reviewed in [12]). Nevertheless, inkjet-based bioprinters have become a massiveusedtechnologyreducingcostsand facilitatingtheuse offreedesignsandsoftwaresworldwide.Currentresearchisnow focusedinthedevelopmentofnoveltechnologiesallowingtheuse ofmultiplecelltypesandmaterials.

Microextrusionsystemsappearedasamodificationofinkjet prin-ters.Heretheextrusionofthematerialtakesplacethrough micro-metricapertures(usuallyaneedlewithinnerdiametersthatrange

from few to hundred micrometers). By applying a continuous

force, this technique allows to print uninterrupted cylindrical

lines. Commonly, these systems robotically extrude biological

materialsby pneumaticormechanical dispensing systemsonto

asubstrate.Ithasbeenreportedthatalmostallkindofhydrogels withvaryingviscosities,aswellasaggregateswithhighcelldensity canbeprintedwiththisapproach(reviewedin[10,12]).Compared toinkjetprinting,microextrusionprintingenablesthedeposition oflargeamountofcells,allowingthegenerationofconstructswith generaltissuesize.Sofar,highconcentrationsofhydrogelssuchas alginate,fibrinandPluronicF-127,amongothers,haveprovedto beeffectivewhenproducingstable3Dcell-ladenstructures[114– 120].Importantly,microextrusionsystemshavebeenparticularly effectivewhenprintingmulticellulartissuespheroidsthatfurther self-assemble into the desired 3D structure (reviewed in [15]).

Although all the advantages described here, one of the major

limitationsofthistechniqueisthedecreasedcellviabilityresulting fromthe shearstresswhencellsareinviscousfluidsduringthe extrusionprocess[121].Thus,oneofthemainchallengesconsists

in the retention of cell viability and printing speed without

decreasingpressureor reducingnozzlesize. Severaltissueshave

been fabricatedusing this system, including branched vascular

trees,aorticvalves,andinvitrotumormodels(reviewedin[12]).

Recently, Atala and coworkers have lead the first work on the

fabricationofbioprintedtissuefunctionalconstructsinvitroandin vivoformandiblebone,ear-shapedcartilageandorganizedskeletal muscleathuman-scale[18].

Inlaser-assistedbioprinting(LABP)dropsofcell-ladenbiomaterials aregeneratedafterlaserpulses.Thefallingbioinkdropletisfurther collectedonthesubstrateandcrosslinked,avoidingshearstressand resultinginhighcellviabilities,evenwhenusinghighlyviscous materials.Thisrecentmethodologyreliesontheuseofalaserpulse

that createsa high-pressure bubbleon a ribbon containing the

materialtobeprinted,therebygeneratingabioinkdroplet.LABPis nozzle-free,thusminimizingclogging-relatedissues.Moreover,the achievedresolutionallowsthedeliveryofsingle-cellsoneachdrop. Sincethissystemgeneratesscaffold-free3Dcellconstructsthrough

a layer-by-layer manner, lately LABP is becoming used for the

deposition of different living cells and biomaterials in a well-defined3Dstructure.Besidestheseadvantages,stillpossibleside

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effectsoflaserexposuretocellsremainelusive.Otherlimitations arerelatedtothepreparationofcell-ladenribbons(specificforeach celltypeandhydrogel,hinderingscaling-upprocedures)andthe depositionofmetallicresiduesinthe finalbioprintedconstruct. Similarly,targetingandpositioningcellsbecomesdifficultbecause ofthenatureoftheribboncellcoating(reviewedin[12]).Todate, fewworkstookadvantageofthissysteminregenerativemedicine

[19,122],andthehighcostofLABPprecludestheiruseformany researchersworldwide.

Stereolithography(SLA)wasinitiallyusedtocreatecell-free scaf-foldsbuttheincreasingdevelopmentintheformulationsofnew cross-linkablematerialsallowedtheuseofSLAfor3Dbioprinting (reviewedin[123])[124].InSLA,thelaserfocusingpointmoveson theX/Yaxesalongtheuncuredbioink,whilethestagewherethe materialis polymerized lowers allowing polymerization. Lately, directlightprojection(DLP)hasemergedasanaffordableand versa-tilevariant ofSLA.InDLP,thelightfromadigitalmicromirror deviceorprojectorisusedforcuringphotocrosslinkablehydrogels in a layer-by-layer approach. By curing the structures

plane-by-plane, DLP offers enormous advantages in front of SLA (e.g.,

printingtimeisnotdependingonthedesigncomplexityofeach plane).DLPhasbeenrecentlyusedforgeneratingbiocompatible

scaffolds[125–127],andmorerecentlyWangandcoworkers dem-onstrated that DLP enabled forthe fabricationof3D cell-laden structureswithresolutionsof50micrometers,andreaching85% cellviabilitywhenencapsulatinghumanfibroblasts[128].DLPand SLAoffernewalternativesforthefabricationof3Dbioconstructs with precisemicro- and nano-architecture, beingaffordable sys-temsintermsofcosts.

Asummary ofthe aforementioned bioprintingtechniques is

presentedinBox6.

3D

bioprinting

of

cell-laden

hydrogels

Since 2000, when rapid prototyping technologies were first

adapted forthe depositionof cell-ladenhydrogel3Dstructures in cell-compatible printing conditions, researchers all over the worldhavetriedtoproducebiologicaltissue-likeconstructsusing

different cell types and hydrogel formulations. The basic 3D

bioprintingequipmentneedswhenaimingtofabricatecell-laden 3DstructuresaredescribedinBox7.

Current effortsaredevoted to developnovel biomaterial

for-mulations thatcanmimic the complexityofthenativeECM–a

conceptcalledbiomimicry–withanimpactforbioprinting appli-cations.ECMcompositionandrigidityhaveprovedtodictatecell

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BOX7

3Dbioprintingofcell-ladentissueconstructs

3Dbioprinterneeds Allowcombinationofmicro-extrusion,fuseddepositionandinkjetprintingatthesametimeduringa printingprocedure

Haveatleasttwohydrogel-compatibleprintingheads,whichfacilitatesdepositionofdifferentcelltypes inthesameprintedconstruct

Possesstemperaturecontrolsystemsforprintingheadsandsubstrate Allowopticalmonitoringoftheprintingprocess

Allowtheinterchangeofmaterialcartridgesduringtheprintingprocedure Precisecontrolofthepressureappliedduringtheprintingprocess

Allowtheuseofdifferentsubstratestoprintthedesired3Dtissueconstruct(e.g.petridish,multiwall plates)

Possesanaccuratecalibrationsystemforneedletippositioning Possesasourceoflight(UV,bluelight)forlight-crosslinkinghydrogels Workundersterileconditions

Keymaterialpropertiesfor3Dbioprinting Printabilitydefinesthesuitabilityofamaterialforaspecificprintingprocessandlargelydependsonthe materialphysicochemicalproperties(viscosity,shearthinning,yieldstress,hydrogelcrosslinking mechanism)undertheconditionsprovidedbythebioprintinginstrument

Biocompatibilityreferstotheabilitytoperformasamaterialthatwillsupporttheappropriatecellular activity,includingthefacilitationofmolecularandmechanicalsignalingsystems,inordertooptimizetissue regeneration,withoutelicitinganyundesirablehostresponses

Biodegradabilitydescribesthebiologicalprocessesinsidethebodythatcauseagradualbreakdownofa material.Degradationkineticsshouldbematchedtothenovotissueformationandthebyproducts generatedshouldbenontoxic

Biomechanicalpropertiesofthematerialshouldprovidesufficientstructuralintegritytotheprinted constructandmatchtissuespecificbiomechanicrequirements

Biomimicryreferstotheabilityofreproducetissue-specificendogenousmaterialcompositions 3Dbioprintingprocessingparameters Thefourmainvariablesthatneedtobebalancedtooptimizetheprintingprocedureandensurethe

designfidelityare:theneedle/tipsize,thedistancefromthetiptothesurface,thematerialflowrateand thelinearwritespeed

Thesevariablesarebalancedwhentheleadingedgeoftheprintedbioinkiscontinualwiththeneedle, beingpossibletodispenseauniformstrand

Theseaforementionedvariablesneedtobeoptimizedforeverydifferentmaterialused

Changesonenvironmentalconditionssuchashumidityandtemperaturemayinfluenceprintingfidelity Asprintedstructuresincreaseincomplexity,theincorporationofsupportstructureswithinthefabricated patternisnecessary

Theprintingtimeshouldbeconsidered,sinceextendedperiodsoftimeatnon-optimaltemperature, humidityormaterialbiocompatibilitywillaffectbothmaterialpropertiesandcellviability

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fateandfunction(reviewedin[129]).Inthehumanbody,tissue rigidityrangesfrom0.2–5kPainsofttissuesasbrain,to15,000kPa

in bone, beingan important parameterto beconsidered when

aimingtodesign3Dtissueandorgananalogues.

Besides the need to be biocompatible and biodegradable, a

biomaterial formulation for bioprinting must possess suitable

physicochemical properties in order to fabricate 3D constructs

withhighresolutionandprintingfidelity–acharacteristicnamed printability(Box7)(reviewedin[130–132]).Inaddition,itmust alsobeoptimizedinordertominimizestress-induceddamageto

the cells and biological components, which occur during the

deposition process (reviewed in [130–132]). The ideal hydrogel

formulationshouldreachacompromisebetweenpreservingcell

viabilityandmatchingoptimalprintability.

Viscosity,shearthinningandyieldstressofadefinedbioinkwill directlyaffectprintingfidelityduringthebiofabricationprocess. Similarly,thespecificprocessingparameterswilldefinetheshear stress that cells willsuffer during the deposition and the time requiredforthefabricationofagiven3Dconstruct(e.g.,tipsize, flowrate,temperature;Box7).Anotherimportantoutcomeisthe maintenanceofsufficientstructuralintegritybythebioprinted3D construct,whichwillbemainlydependentonthegelationofthe

hydrogelformulation.Naturalandsynthetichydrogelsappliedto

3Dbioprintingandtheircorrespondentgelationmechanismare

furtherreviewedelsewhere(reviewedin[130–133]).

Elegantworkshavedemonstratedthefeasibilityof3D bioprint-ingforthegenerationofseveraltissues,includingbone[18],skin

[19],vasculargrafts[118,120,134],trachealsupports[135],heart

[20,117,136]and cartilage[18,20]tissuestructures,usingseveral cellsourcessuchashumanumbilicalveinendothelialcells,

mes-enchymal stem cells, human meniscus cells and fibroblasts

(reviewedin[10,12]).However,thefabricationofhumansizeorgan analogueswithcomplexarchitecturesrequiresamoreelaborate3D bioprintingstrategy,usuallyinvolvingtheco-depositionofother materialcomponents(suchassupportiveandsacrificialmaterials), togetherwith cell-ladenhydrogels,ensuringsufficient structural integritytothe printedconstructwhile maintaininganinternal porosity (Fig. 3). In this regard, the recent work by Atala and coworkersprovidesa3Dbioprintingplatformfortheproduction oftissuesforhumanapplications,pavingthewayforfuture build-ingofsolidorgans[137].

Acellular

organ-specific

dECM

hydrogels

for

3D

bioprinting

As decellularization protocols emerged, hydrogels made from

decellularized tissues including urinary bladder [138], heart

[139],liver[140],dermis[141],adiposetissue[142],bone[143], and lung[144],among others,weredevelopedand reportedto supportgrowthandfunctionofdifferentcelltypes.However,itis notuntilveryrecentlythattissue-specificdECMhydrogelshave

beenenvisionedasa newclassofhydrogelsfor3D bioprinting

[20,145–147].Nowadays, one of the main hurdles when using

dECM hydrogelsasbioinks relieson their lowviscosity, which

inevitablycompromise shapefidelityofthebioprinted3D

con-struct,worseningprintingresolution.

To date,only fewstudies have used dECM hydrogels for 3D

bioprinting,applyingdifferent strategies toimprove their print-ability[20,145–147].Patiandcoworkerswerethefirstto

success-fully apply dECM hydrogels for 3D bioprinting. Their strategy

consisted on the co-deposition of an open porous structure of

polycaprolactone (PCL) as a supportive material, together with

thecell-ladendECMhydrogelmadefromcartilage,heartoradipose porcinetissues[20].Recently,thesamegroupusedskeletal muscle-deriveddECMhydrogelsfor3Dprintingofmuscleconstructs[147]. Followingadifferentstrategy,Skardalandcoworkerselegantly

developedmodularhyaluronicacidandgelatin-basedhydrogels

supplemented with porcine liver, cardiac and skeletal muscle

dECM solutions. Following a two-step crosslinking procedure,

the authors achieved printable bioinks with different stiffness rangingfrom100Pa to20kPa, thus allowingthe possibilityto mimicthe mechanical characteristics ofdifferent tissuesin the body[145].OtherrecentworkbyJangandcoworkerstook advan-tageofpigheartdECMtopreparecardiac-specifichydrogelsthatin

combinationwithhumancardiac progenitorcellswereusedto

fabricate3Dbioprintedcardiacconstructs.Thegelationofcardiac

dECMhydrogelswasbasedonthermalandchemicalcrosslinking

usingvitaminB2viaUVAactivation[146].

Future

outlook

Amajorlimitationwhengeneratingartificialorgansondemand

standsinthedevelopmentoftechniquestoproperlyreintroduce

FIGURE3

Schematicrepresentationofa3Dbioprintingsystemconsistingofa computeraided3-axisstagecontrollerandadepositionmoduleincluding threedifferentprintheadsconnectedtoapressurecontroller(a).Computer aideddesignandcomputeraidedmanufacturing(CAD/CAM)processfor 3Dbioprintingofahumansizekidney.A3DCADmodelgeneratedfrom medicalimagingdata(CT:computedtomography;MRI:magneticresonance imaging)producesavisualizedmotionprogramwhichdictatestheXYZ stagemovementstogeneratethe3Dbioprintedkidneyprototype(b).

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cellsintotheorgan-specificdECMscaffolds,assessingboth

com-plete organ re-endothelization and functional activity. Due to

theirintrinsiccharacteristics,hPSCshavebeenenvisionedasan optimalcellsourceforthegenerationofcomplextissuestructures liketheorganparenchymaandthevascularsystem,offeringmajor

advantageswhencomparedwithadultsomaticor stemcellsfor

thesamepurposes.Moreover,targetedgenomeediting,asCRISPR

platform, is a powerful tool to manipulate and correct disease

relatedgenesinpatient-derivedhiPSCs,allowingforthe genera-tionof autologous-correctedcellssuitable fordisease modeling anddrugscreening[148].Webelievethattargetedgenomeediting

approachescombined with recentprogressin the formation of

patient-specific hiPSCs-derived organoids could provide an

un-precedentedsource of organ-specific cell types suitablefor cell

replacement therapies. In this regard, common efforts on the

definitionofchemicallydefined conditions toculture

hPSC-de-rived organ-specific cells has led to the examination of novel

approachesguidinghPSCsmaturation.Followingthesequestions, differentworkshaverelayedontheuseofhumandECMscaffolds

together with hPSCs in order to generate human tissue grafts

[43,84,104],and even toreconstruct wholeorgans [84,105], re-vealingtheimpactoforgan-deriveddECMsontheproper instruc-tionofhPSCsfateandfunction[40,43,84].

Alternatively,3Dbioprintingrepresentsaformidable technol-ogyforartificialorgangeneration.Besidesthedifferentlimitations of this nascenttechnology (e.g., printing resolution and time, combinationofdifferentbioinkssimultaneously,amongothers),

the possibility to print human-scale tissues has been recently

demonstrated [18].Inthis regard,seminal studies havealready provedthe feasibilityto print3Dtissue constructsusing

organ-specific dECM hydrogels as biomimetic bioinks [20,145–147],

openingthedoortothefabricationofnovelbioinkformulations

matching cytocompatibility and mechanical strength

require-mentsfor3Dbioprinting.

Overall,webelieve that recentadvancementsinthe fieldsof hPSCsdifferentiationtogetherwithorgan-deriveddECMscaffolds

ornoveldECM-basedhydrogelsaimedfor3Dbioprinting

repre-senta stepforward in the fabrication ofautologous functional tissueson-demand(Fig.4andBox8).Tothisend, multidisciplin-aryresearchinthefieldofengineering,biomaterialsscience,stem

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FIGURE4

3Dbioprintingapproachforthedevelopmentofpatient-specificorgananaloguessuchaskidney.Patient-specificiPSCs(a)aredifferentiatedintorenal progenitorcellsin2Dmonolayercultures(representativeimageforSALL1andWT1doublepositiverenalprogenitorsbyimmunofluorescence)(b)thatare furtherinducedtodevelopintokidneyorganoids(representativeimageforPAX8andE-cadherinpositive3Drenal-likestructuresbyimmunofluorescence) (c).ThecombinationofkidneydECM-basedhydrogels(d)withpatientiPSC-derivedrenalprogenitorcellscouldbeusedforthebiofabricationofspecialized 3Drenalconstructs(representativeimageforPAX8renalstructuresdevelopedintoprintedkidneydECM-basedhydrogels)(e).Thisapproachrenderstothe generationoftissueandorgananaloguessuitableforregenerativemedicineapplications(e.g.,diseasemodeling,drugscreening,cellreplacementtherapies, amongothers).

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cellbiologyandmedicinewillbeessentialtofurthersucceedinthe biofabricationofautologousorgansforfutureclinicalreplacement strategies.

Acknowledgements

WethankSOLIDCAMSTUDIOfor3Dmodelling,printingand

rendering.E.GwassupportedbyStG-2014-640525_REGMAMKID.

R.OwasfoundedbyaFIfellowship(Secretariad’Universitatsi

RecercadelDepartamentd’EconomiaiConeixementdela

Generalitatde Catalunya).C.Twas supportedby

StG-2014-640525_REGMAMKID. P.PwaspartiallysupportedbyMINECO

(SAF2014-59778). F.F.A.wassupportedbyInstitutode Salud

CarlosIII-ISCII(MINECO:PI10-00141andPI10-02038),Redde

Investigacio´n Cardiovacular(RIC)andRedTerCelfrom ISCII

(MinistryofEconomyandCompetitiveness,Spain),andCAM:

S2010/BMD-2420andhasbeenfinanciallysupportedby the

CommissionforUniversitiesandResearchoftheDepartmentof

Innovation,Universities, andEnterpriseoftheGeneralitatde

Catalunya(2014 SGR1442)anddevelopedinthecontextof

ADVANCE(CAT)withthesupportofACCIO´ (CataloniaTrade&

Investment;Generalitatde Catalunya)andtheEuropean

Community undertheCatalonianERDF operationalprogram

(EuropeanRegionalDevelopmentFund)2014-2020.This

workalsowaspartially supportedbytheprojectMINDS

(TEC2015-70104-P),awarded bytheSpanish Ministry

ofEconomyandCompetitiveness,andbyCERCA

Programme/GeneralitatdeCatalunya.N.M wassupportedby

StG-2014-640525_REGMAMKID,MINECO, (SAF2014-59778,

RYC-2014-16242),andbyCERCAProgramme/Generalitatde

Catalunya,andCardioCel(TerCel,InstitutodeSaludCarlosIII)

and2014SGR1442.

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Challengesandfutureperspectives

Researcharea Specificchallenges Futureoutcomes Decellularization Standardizationofdecellularizationprotocolsforeach

specificorgan

Systematicevaluationofimmunologicresponseandthe possibilityofusingdECMscaffoldsfromdifferentspecies

Robustproductionoforgan-deriveddECMscaffolds Determinationoftheneedofimmunosuppressivedrugs aftertransplantation.Increasethetissuesource,overcoming donorshortage

Recellularization Improvedseedingmethodologiesforcomplete re-endothelizationoftheorganvascularbedandparenchyma recellularization

Novelbioreactorsystemswithoptimalcontroloverthe cultureconditions(oxygendelivery,biophysicalstimulation, continuousmonitoringoffunctionalparameters)

Optimalvascularization

Determinationoforganbiochemical/biophysical parametersduringrecellularization,andend-pointsforeach organ

Definitionofcommonguidelinesfortheassessmentof organmechanicsPreservationofbioengineeredorgans beforetransplantation

Functionalevaluationinlargeanimalmodels Humanpluripotent

stemcells

Standardizedprotocolsforexpansionanddifferentiationto largecellnumbers

PurificationofhPSC-differentiatedcellsandidentification ofoptimalcellcultureconditions(growthfactors,cytokines) forcellphenotypemaintenance

IdentificationofhPSC-derivedprogenitorcelltypessuitable forengraftmentandmaturationintodECMscaffolds

Repopulationoforgan-deriveddECMscaffoldswith multiplehPSC-derivedprogenitorcelltypesspecificforeach organcompartment,andinsufficientcellnumbertoachieve comparablenativeorgancelldensities

Generationofpersonalizedhumanbioengineeredorgan equivalentswithoptimalfunctionalactivity

3Dbioprinting Developmentofnovelcell-compatiblehydrogelswith tailoredviscosityandbiochemicalcomposition

Monitoringofbioinkflowratewithautomaticadjustment ofprintingconditions

Presentalignmentcapabilityonpreviouslyprinted constructs

Improvebioprintingfullautomation

Developmentofbioreactorsfortissue/organgrowthand maturationpost-processing

Bioprintingofcell-ladenstructureswithincreased mechanicalstrength,bioactivityandcellviability Increasedprintingfidelity

Step-wisefabricationofcomplexmulticellularstructures Rapidproductionof3Dprintedconstructs,thereby improvingcellviability

Generationoffunctionaltissue/organs

12

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