history and applications of hydrogels · in many applications,a functionaladditiveis blended into a...

2015iMedPub Journalshttp://www.imedpub.com Vol. 4 No. 2:13

Journal of Biomedical SciencesISSN 2254-609X

Review Article

DOI: 10.4172/2254-609X.100013

© Under License of Creative Commons Attribution 3.0 License | This Article is Available in: www.jbiomeds.com 1

Naziha Chirani1,L’Hocine Yahia1,Lukas Gritsch1,2,Federico Leonardo Motta1,2,Soumia Chirani3 andSilvia Faré2

1 Laboratoired’innovationetd’analysedeBioperformance,Ecolepolytechnique,UniversitédeMontréal,Montréal,Quèbec,Canada

2 DipartimentodiChimica,MaterialiedIngegneriaChimica"G.Natta",PolitecnicodiMilano,Edificio6,piazzaL.daVinci32,20133Milano(Italy)

3 LaboratoiredeChimieOrganique,Physiqueetmacromeléculaire,Départementdepharmacie,Facultédemédecine,UniversitéDjilaliLiabés,SidiBel-Abbès,Algeria

Corresponding author: L’HocineYahia

Laboratoired’innovationetd’analysedeBioperformance,Ecolepolytechnique,UniversitédeMontréal,Montréal,Quèbec,Canada

[email protected]

Citation: ChiraniN,YahiaLH,GritschL, etal.HistoryandApplicationsofHydrogels.JBiomedicalSci.2016,4:2.

IntroductionHydrogels are polymericmatrixes that swell but don’t dissolve(intheshortterm)inwater[1].Theswellingpropertiesareduetothehighthermodynamicalaffinitythatthisclassofmaterialshas for the solvent itself. In the past years this characteristic,coupledwithahighversatilityandahightunabilityofmaterial’sproperties,leadtodeepresearchandexploitationofhydrogels.These networks establish equilibrium with the liquid andtemperature of their surroundings for shape and mechanicalstrength. Variations in the concentration, structure and/orfunctionality of themonomer and/or cross-linker used in suchgels can change the structure. Indeed, many new gel-formmaterials,withaplethoraofaimsweredevelopedandtestedindifferent fields of engineering (e.g. environmental, electronics,biomedical),biotechnologyandotherdisciplines.

Thisphenomenoncanbeappreciatedbyanalyzingthenumberofarticlesonthetopic:doingaquickqueryfortheword“hydrogel”onPubMeditiseasytoseetheclearexponentialtendencyinthenumberofarticles(Figure 1).Thisisageneraltrendsharedbyanykindof scientificpublication that is,moreover,underestimatedbecause of the inability of any database to capture thewholescientificliterature.Thisphenomenonisconsiderednotconsistentbymanyacademicpersonalitiesand,surely,generatesconcerns

abouttheabsenceofarealcorrelationbetweenpublishingratesandknowledge[2].Nevertheless,itispossibletoassertthat,evenifthiscourseofeventsisnotevidence,itisatleastaclueofthegrowinginterestofthescientificcommunityonthehydrogeltopic.

Smarthydrogelsystemswithvariouschemicallyandstructurallyresponsive moieties exhibit responsiveness to external stimuliincluding temperature, pH, ionic concentration, light,magneticfields, electrical fields and chemicals. Polymers with multipleresponsive properties have also been developed elegantlycombining two ormore stimuli-responsivemechanisms. Smartpolymer hydrogels change their structural and volume phasetransition as a response to external stimuli resulting in anenormous potential for scientific observations and for variousadvancedtechnologicalapplications.

History and Applications of Hydrogels

AbstractHydrogelshaveexistedformorethanhalfcentury,providingoneoftheearliestrecords of crosslinked hydroxyethyl methacrylate (HEMA) hydrogels. Today,hydrogelsstillfascinatematerialscientistsandbiomedicalresearchersandgreatstrides have been made in terms of their formulations and applications. As aclassofmaterial,hydrogelsareunique,theyconsistofaself-supporting,water-swollenthree-dimensional(3D)viscoelasticnetworkwhichpermitsthediffusionandattachmentofmoleculesandcells.However,hydrogelshaverecentlydrawngreatattention foruse inawidevarietyofbiomedicalapplicationssuchascelltherapeutics, wound healing, cartilage/bone regeneration and the sustainedreleaseofdrugs.This isduetotheirbiocompatibilityandthesimilarityoftheirphysicalpropertiestonaturaltissue.Thisreviewaimstogiveanoverviewofthehistoricandtherecentdesignconceptofhydrogelsandtheirseveralapplicationsbasedontheoldandthemostrecentpublicationsinthisfield.

Keywords: Hydrogel;Applications;Proteomics;Woundhealing;Tissueengineering;Drugdelivery;Vaccine;injectablehydrogel

Received: September01,2015; Accepted: September20,2015; Published: October02,2015

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Hydrogels can be classified into two distinct categories, thenatural and the synthetic hydrogels. Natural hydrogels includecollagen, fibrin, hyaluronic acid, matrigel, and derivatives ofnaturalmaterialssuchaschitosan,alginateandskillfibers.Theyremainthemostphysiologicalhydrogelsastheyarecomponentsoftheextracellularmatrix(ECM)in vivo.Twomaindrawbacksofnaturalhydrogels,however,maketheirfinalmicrostructuresandpropertiesdifficulttocontrolreproduciblybetweenexperiments.First, the fine details of their mechanical properties and theirdependenceonpolymerizationorgelationconditionsareoftenpoorly understood. Second, due to their natural origin (bovinefibrinogen, rat tail collagen… their compositionmay vary fromonebatchtoanother.

In contrast, synthetic hydrogels such as poly (ethylene glycol)diacrylate, poly(acryl amide), poly(vinyl alcohol) are morereproducible,althoughtheirfinalstructurecanalsodependonpolymerization conditions in a subtle way, so that a rigorouscontrolof thepreparationprotocol, including temperatureandenvironment control, may be necessary. Generally speaking,synthetic hydrogels offer more flexibility for tuning chemicalcompositionandmechanicalproperties;userscan,forexamplevarytheconcentrationormolecularweightoftheprecursor,oralterthepercentageofcrosslinkers.Theycanalsobeselectedortunedtobehydrolysableorbiodegradableovervariableperiodsoftime.

Hydrogelsmay be chemically stable or theymay degrade andeventuallydisintegrateanddissolve.Theyarecalled«reversible»or «physical» gels when the networks are held together bymolecular entanglements, and/or secondary forces includingionic, H-bonding or hydrophobic forces (Figure 2) [3]. Physical

hydrogels are not homogeneous, since clusters of molecular entanglements, or hydrophobically-or ionically-associateddomains,cancreateinhomogeneities.Freechainendsorchainloopsalsorepresenttransientnetworkdefectsinphysicalgels.

Whenapolyelectrolyteiscombinedwithamultivalentionoftheopposite charge, itmay form a physical hydrogel known as anionotropichydrogel,calciumalginateisanexampleofthistypeof hydrogel. Hydrogels are called “permanent” or “chemical”gelswhentheyarecovalently-cross linkednetworks (Figure 3). The synthetic hydrogels of Wichterle and Lim were based oncopolymerizationofHEMAwiththecrosslinkerEGDMA.Chemicalhydrogels may also be generated by crosslinking of water-solublepolymers,orbyconversionofhydrophobicpolymerstohydrophilicpolymerspluscrosslinkingisnotnecessary.Insomecases,dependingonthesolventcomposition,temperatureandsolidsconcentrationduringgelformation,phaseseparationcanoccur,andwater-filledvoidsormacroporescanform.Inchemicalgels,freechainsendsrepresentgelnetwork“defects”whichdonot contribute to the elasticity of thenetwork.Other networkdefects are chain loops and entanglements,which also do notcontributetothepermanentnetworkelasticity.

In many applications, a functional additive is blended into apolymermatrix to enhance its properties. However, when thepolymer and functional additive are applied to a surface, thefunctional molecule may be easily lost. In favorable cases, itmay be possible to incorporate the additive directly into thepolymer as a comonomer. In recent study, a functionalizedpolymerhasbeenobtainedthroughthecombinationof linkingaphotodynamic,antimicrobialdye,RoseBengal,tovinylbenzylchloride via etherification and then polymerizing this into a

Figure 1 Histogramshowingtheincreaseinpublicationsrelatedtothekeyword“hydrogel”duringthepast45years.Aproperexponentialfittingisalsodetectable.

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3© Under License of Creative Commons Attribution 3.0 License

water-solublepolymerusingchaingrowthcopolymerization[4].TheamountofunincorporateddyewasdeterminedusingdialysisinbuffersolutionatpH=7,4.WhenRoseBengalwasfirstreactedwith vinyl benzyl chloride before polymerization, it was foundthatitwasnearly100%incorporatedintothecopolymer.

ReviewHistoryTheword“hydrogel”,accordingtoLee,KwonandPark,datesbacktoanarticlepublished in1894.Anyway, thematerial describedtherewasnotahydrogelaswedescribeittoday;itwasindeedacolloidalgelmadewithinorganicsalts.Isyetremarkabletonoticehowthehistoryofthetermitselfisconsistentlylong[5].Anyhow,thefirstcrosslinkednetworkmaterialthatappearedinliteratureandhasbeendescribedbyitstypicalhydrogelproperties,oneforall thehighwater affinity,was apolyhydroxyethylmethacrylate(pHEMA) hydrogel developed much later, in 1960, with theambitiousgoalofusingtheminpermanentcontactapplicationswith human tissues, hydrogels are in fact the first materials

developedforusesinsidethepatient[6,7].Sincethenthenumberofstudiesabouthydrogelsforbiomedicalapplicationsbegantorise,especiallyfromthedecadeof70's[5].Theaimsandgoalsand thenumberofmaterials changedandenlarged constantlyovertheyears.AssuggestedbyBuwaldaetal.[8],thehistoryofhydrogelscanbedividedinthreemainblocks.

A hydrogel’s first generation that comprises a wide range ofcrosslinkingprocedures involving thechemicalmodificationsofamonomerorpolymerwithan initiator. Thegeneral aim is todevelopmaterialwithhighswelling,goodmechanicalpropertiesandrelativelysimplerationale.

Then, starting in the seventies, adifferent conceptofhydrogelgrewinimportance:asecondgenerationofmaterialscapableofaresponsetospecificstimuli,suchasvariationsintemperature,pHorconcentrationofspecificmoleculesinsolution.Thesespecificstimuli can be exploited to trigger likewise specific events, forexamplethepolymerizationofthematerial,adrugdeliveryoranin situporeformation[9].

Finally, a third generation of hydrogels focusing on the

Figure 2 Typesofcrosslinking,adaptedfromA.S.Hoffmann,“AdvancedDrugDeliveryReviews”[29],ontheleftofthefigureitispossibletoseethechemicalcrosslinkednet,ontherightthephysicalone.

Figure 3 SDS-PAGEmigrationforseparationofproteinsmixture[87].

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investigation and development of stereo complexed materials(e.g.PEG-PLAinteraction)[10,11]hydrogelscrosslinkedbyotherphysicalinteractions(e.g.cyclodextrines)[12,13].

This progress in hydrogel’s science is quickly leading to anincreasing interest in the development of the so called “smarthydrogels”, polymeric matrixes with a wide spectrum oftunablepropertiesandtriggerstimuli.Thetopic istheoreticallyinexhaustible and the possible applications, the engineeringandmedicaldevicesthatcanbeobtainedfromitareaboveanyimagination.

Since the pioneering work of Wichterle and Lim in 1960 oncrosslinked hydrogels [7], and because of their hydrophiliccharacterandpotentialtobebiocompatible,hydrogelshavebeenofgreatinteresttobiomaterialscientistsformanyyears[14-16].

TheimportantandinfluentialworkofLimandSunin1980[17]demonstrated the successful application of calcium alginatemicrocapsulesforcellencapsulation.Laterinthe1980s,Yannasand coworkers [18] incorporated natural polymers such ascollagen and shark cartilage into hydrogels for use as artificialburndressings.Hydrogelsbasedonbothnatural and syntheticpolymers have continued to be of interest for encapsulationof cells [19] and most recently such hydrogels have becomeespeciallyattractiveto thenewfieldof“tissueengineering”asmatricesforrepairingandregeneratingawidevarietyoftissuesandorgans[20].

Physical and chemical propertiesDespite much progress, a fundamental understanding of gelpropertiesisnotyetsufficientforarationaldesignofnovelgelsystems. For such designs, it is important to know how solutemoleculesinteractwiththegel,inparticular,howtheypartitionbetween the gel phase and the surrounding liquid phase.Partitioning depends on two major effects: size exclusion andmolecularattraction/repulsion.

Swelling: Hydrogels are cross-linkedpolymernetworks swollenin a liquid medium. The imbibed liquid serves as a selectivefiltertoallowfreediffusionofsomesolutemolecules,whilethepolymernetworkservesasamatrixtoholdtheliquidtogether.Hydrogelsmayabsorbfrom10-20%(anarbitrarylowerlimit)uptothousandsoftimestheirdryweightinwater.

The character of the water in a hydrogel can determine theoverall permeation of nutrients into and cellular products outof the gel. When a dry hydrogel begins to absorb water, thefirstwatermoleculesenteringthematrixwillhydratethemostpolar, hydrophilic groups, leading to primary bound water. Asthepolargroupsarehydrated,thenetworkswells,andexposeshydrophobic groups,whichalso interactwithwatermolecules,leading to hydrophobically-bound water, or secondary bound water.Primaryandsecondaryboundwaterareoftencombinedand simply called the total bound water. After the polar andhydrophobic sites have interacted with and bound watermolecules, the network will imbibe additional water, due totheosmoticdrivingforceofthenetworkchainstowardsinfinitedilution. This additional swelling isopposedby the covalentorphysicalcrosslink,leadingtoanelasticnetworkretractionforce.

Thus, thehydrogelwill reachanequilibriumswelling level.Theadditional swellingwater that is imbibed after the ionic, polarandhydrophobicgroupsbecomesaturatedwithboundwateriscalledfreewater orbulkwater,andisassumedtofillthespacebetweenthenetworkchains,and/orthecenteroflargerpores,macroporesorvoids.Asthenetworkswells,ifthenetworkchainsorcrosslinkaredegradable,thegelwillbegintodisintegrateanddissolve,ataratedependingonitscomposition.

Thereareanumberofmethodsusedbyresearcherstoestimatetherelativeamountsoffreeandboundwater,asfractionsofthetotalwatercontent.Allofthemarecontroversial,sincethereisprotonNMRevidence that the interchangeofwatermoleculesbetweentheso-calledboundandfreestatesisextremelyrapid,perhapsasfastasoneH2Omoleculeevery10-9s.Thethreemajormethodsused to characterizewater inhydrogels arebasedontheuseofsmallmolecularprobes,DSCandNMR.Whenprobemolecules are used, the labeled probe solution is equilibratedwiththehydrogel,andtheconcentrationoftheprobemoleculein the gel at equilibrium ismeasured. Assuming that only thefree water in the gel can dissolve the probe solute, one cancalculatethefreewatercontentfromtheamountoftheimbibedprobe molecule and the known (measured) probe moleculeconcentrationintheexternalsolution.Thentheboundwaterisobtainedbydifferenceof themeasured totalwater contentofthehydrogelandthecalculatedfreewatercontent.

The use ofDSC is basedon the assumption that only the freewater may be frozen, so it is assumed that the endothermmeasuredwhenwarmingthefrozengelrepresentsthemeltingofthefreewater,andthatvaluewillyieldtheamountoffreewaterintheHGsamplebeingtested.ThentheboundwaterisobtainedbydifferenceofthemeasuredtotalwatercontentoftheHGtestspecimen,andthecalculatedfreewatercontent.

In another formulation, swelling is the property to absorbwaterandretain it forarelative longtime. Itcanbeevaluatedby measuring the dry weight and the swollen-state weightand computingeither aponderal variation (wateruptake)or avolumeofadsorbedsolvent(boththequantitiesareconsideredaspercentages).

[ ]. . 100 1−= ×

swollen weight dry weightW U dry weight

[ ]. . . 100 2swollen weight dry weightV A S

water density−

= ×

As simple as it may seem, the evaluation of swelling is theprincipalassay tobeperformedonhydrogel samples,as it canbeameasureformanyoftheirproperties:crosslinkingdegree,mechanical properties, degradation rate and so on. For manygels, the evaluation of swelling and swollen state stability isthesimplest,cheapestandsurestwaytodiscriminatebetweencrosslinkedgelsandthenotcrosslinkedoriginalpolymer[21].

Mechanical propertiesThemechanicalpropertiescanvaryandbetuneddependingonthepurposeof thematerial. It is possible toobtain a gelwithhigherstiffnessincreasingthecrosslinkingdegreeorloweringitby heating thematerial. The changes inmechanical propertieslinktoawiderangeofvariablesandcausesanddifferentanalysis

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mustbemadeaccordingtothematerial,theconditionsandtheaimof thestudy.Forexample,whilegelatinshowanoticeableincreaseinYoungModulusthroughcrosslinking[22],silkfibroinhas a very high Young Modulus, but after the revitalization itwill decrease [23]. These properties (young modulus, Poissonmodulus, storage and loss moduli, tanδ) can be evaluated bya DynamicMechanical Analysis (DMA) device or a rheometer,accordingtothethousandsoftechniquesavailableonthemarketthatwillbenofurtherdiscussedhere[24,25].

It’s importanttonotethat inahydrogel, theYoungModulus isthe results of the union between water and gel matrix. If wehavetoseedsosteoblastcellswewillneedamorestiffmaterialthanifwecultureadipocyte,thesamerationaleisvalidforthedevelopmentofaheterogeneousprostheticdevice,forexamplesubstitutefortheintervertebraldisc.

Porosity and permeation: Poresmaybeformedinhydrogelsbyphaseseparationduringsynthesis,ortheymayexistassmallerporeswithin thenetwork.Theaverageporesize, theporesizedistribution,andtheporeinterconnectionsareimportantfactorsofahydrogelmatrixthatareoftendifficulttoquantify,andareusually included together in the parameter called « tortuosity». The effective diffusion path length across a HG film barrieris estimated by the film thickness times the ratio of the porevolumefractiondividedbythetortuosity.Thesefactors,inturn,aremostinfluencedbythecompositionandcrosslinkdensityofthehydrogelpolymernetwork.

Labeledmolecularprobesofarangeofmolecularweights(MWs)ormolecularsizesareusedtoprobeporesizesinhydrogels[26].

Pore-sizedistributionsofhydrogelsarestronglyaffectedbythreefactors:

· Concentrationofthechemicalcross-linksofthepolymerstrands. That concentration is determined by the initialratioofcross-linkertomonomer.

· Concentration of the physical entanglements of thepolymerstrands.Thatconcentrationisdeterminedbytheinitialconcentrationofallpolymerizablemonomersintheaqueoussolution.

· Net charge of the polyelectrolyte hydrogel. That chargeisdeterminedbytheinitialconcentrationofthecationicand/oranionicmonomer.

Thesethreefactorscanbequantifiedusingthecompositionofthehydrogel,thatis,bythenominalconcentrationsofmonomerandcross-linker:

The porous structure of a hydrogel is also affected by theproperties of the surrounding solution, especially by dissolvedionicsolutes(Donnaneffects)andbydissolvedunchargedsoluteswhichpartitionunevenlybetweenthegelphaseandthesolutionphase(Osmoticeffects).

Forrationaldesignofhydrogels,itisusefultoknowthepore-sizedistributiondependsonthehydrogelcharacterizationcommonly

expressedby%Cand%T.

Most techniques used to investigate the porosity of hydrogelsare limited because they require the pore solvent and/ortemperature to be altered, causing the gel to shrink, swell orrequiremathematicalmanipulationandassumption,whichmayintroduceunwantedartifacts.

Porosity is a morphological feature of a material that can besimplydescribedasthepresenceofvoidcavityinsidethebulk.Itisusefultocontroltheporosityinmanydevicesforawidevarietyofapplications,suchasoptimalcellmigrationinhydrogel-basedscaffoldsortunablelode/releaseofmacromolecules.

( ) [ ]100 5= ⋅+pore

bulk pore

VPorosity %

V VIn a sample, pores can show differentmorphologies: they can beclosed,openasablindendorinterconnected,againdividedin cavities and throats. These porosities can had been studiedand evaluated in papers in the past decades using a variousspectrumoftechniques.Firstofall,porositycanbeevaluatedbytheoreticalmethods,suchasunitcubeanalysis,masstechnique,Archimedesmethod,liquiddisplacementmethod.Theseanalysisarecommonlycoupledwithopticalandelectronicmicroscopy.

Otherinterestingtechniquesarethemercuryporosimetry,basedon Washburn’s equation, with the inconvenience of being adestructiveassay,thegaspycnometry, thegasadsorption(thatcanbeissuedusingdifferentproceduressuchassmallquantityadsorption, monolayer and multilayer adsorption), liquidextrusionporosity,anassaythatpermits toevaluatedsample’spermeability too, capillaryflowporosity, again a test basedonWashburn’s equation. Furthermore another important assay istheMicro-CT,alsocalledX-rayMicrotomography,arelativenewimagingtechnique,simplydescribedasanon-destructivehigh-resolution radiography, capable of qualitative and quantitativeassaysonsamplesandevaluationoftheirporeinterconnections.Betweenthequantitativeassaysthatcanbeperformed,micro-CTcangiveinformationonaverageporesize,poresizedistribution,pore interconnection, struts/walls thickness and anisotropy/isotropy of the sample (in the sense of presence/absence ofpreferential orientation of the pores). It is yet, nowadays, anexpensivetechniquebothintermofmoneyandtime[27,28].

Microscopy techniques can be used in thousands of differentassaysinvolvinghydrogels.Theyarebothinvolvedinqualitativeand quantitative tests, from simplemorphological assessing ofmaterial’spropertiestomorecomplexbiocompatibilityassays.

Briefly, by microscopy techniques topography and surfacemorphology canbeassessed.These techniques canbedividedin many classes, by increasing magnification power: opticalmicroscopy(OM),stereomicroscopy(SM),electronmicroscopy(SEM and TEM), tunneling microscopy (STM), atomic forcemicroscopy(AFM)[29].

Crosslinking: Crosslinkingisnotproperlyapropertyofhydrogels,whileitismoreofacauseofalltheotherpropertiesofthematerialitself.Thecrosslinkingdegreecanbecorrelatedtobasicallyeverycharacteristicof a hydrogel. Thenatureof the crosslinking canvary a lot. Indeed, the hydrogel’s network can be obtained in

[ ]4weight of x crosslinker%C weight of monomer weight of x crosslinker

−=

+ −

[ ]3weight of monomer weight of x crosslinker%T total volume

+ −=

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manydifferentways.Theprocessescanbedividedintotwobigcategories:firstofallthesocalledphysicalcrosslinkingthatoccurs,forexamplethankstohydrophobicinteractionsbetweenchains,ionicinteractionsbetweenapolyanionandapolycation(complexcoacervation) or ionic interactions between a polyanion andmultivalent cations (ionotropic hydrogel). The second categorycomprises the chemicalboundgels. The crosslinking canoccurby ultraviolet irradiation, heating or chemical crosslinking viacrosslinkerwithahugeensembleofreactions,suchasMichael’sreaction,Michaelis-Arbuzov reaction, nucleophile addition andsoon[30].

By controlling the degree of crosslinking is possible to tunethepropertyof thematerialandoptimize it formanydifferentapplicationsgettingtheoretically,inthisway,awidespectrumofapplicationsstartingfromthesameoriginalpolymer[33,34].

Applications: From the Lab to the Market

Hydrogels arewidely used in the area of electrophoresis, bio-separations, proteomic, chromatography, tissue engineering…etc.Theyarewell-knowninfoodsandmedicinesasabsorbentsin disposable diapers, as filters for water purification and asseparation materials for chromatography and electrophoresis.They are also of interest for controlled drug release and forconcentrationofdilutesolutionsofmacromolecules[35].

Domestic usesIt’s notmandatory for life changing inventions to be high-techdevices with out-of-sight ambitions. Sometimes the simplestexpedient can lead to a bigger progress than expensive andfuturistictechnologies.Thisistrueforhydrogels,too.Thisclassofmaterialshasplentyofdomesticusagesthatexploittheirabilityofloading,retainingandreleasingfluidstosignificantlyimproveeverydaylife.

Diapers: Aninterestingapplicationofhydrogel’sthermodynamicalaffinityforwater,asnotfancyasitcanbe,istheproductionofsuper-adsorbentdiaperswiththepropertyofbeingdryevenafteraconsiderableadsorptionoffluids.Thisisdueto,aspreviouslysaid,thenatureofhydrogel’swateradsorption:thesematerialsdon’tactassponges,unstablytrappingliquidsintotheirpores,butinsteadtheyretainwater(or,sometimes,othersolvents)inwhileiftheyarecarryingconsiderablequantitiesofwateratthesametime.Thedevelopmentofhydrogel-containingdiapers,mostofthemloadedwithdifferentformulationsofsodiumpolyacrylate[36-38], in the past twodecades cut downon a huge numberof dermatological conditions related to a prolonged contactwith wet tissues. Nevertheless many health concern aroseonthe massive usage of disposable diapers: ≈95% of nappies inwesterncountriesaredisposableonesandopinionsaboutclothnappiesarestillconflicting[39].Indeed,manychemicalsusedintheproductionofsuchproducts,likescents,leak-proofmaterialsandsuper-adsorbentpolymers,seemtobekeyelementsforthedevelopmentofmanyconditions,fromchronicdiaperrashandasthma,tomoreseriousproblemssuchasmaleinfertilityoreventesticularcancer.Furthermore,disposablediapers,sincetheyareusedinhugequantity,createanotableenvironmentalissuesinceit’snoteasytodisposeofthem[40].However,thisisatopicthat,

asfarasinterestingandimportant,goesbeyondthepurposeofourreview.

Watering beads for plants: Another simple application ofhydrogels consists in rough powders of polyacrylamide orpotassiumpolyacrylatematrix soldwithahuge rangeofnames(Plant-Gel,SuperCrystals,Water-GelCrystals)andusedas longtermreservoirofwaterforplantgrowthingardening,domesticand sometimes industrialhorticulture.On theopposite sideastheoneofdiaper’shydrogel, thesematerialsareoptimized fortheirabilityofreleasingwater,insteadoftheabilityofretainingit.Thesustainedreleaseofmanydiversespeciesis,indeed,oneofthemainstrengthofhydrogelsonthemarket,fromgardeningto genetic engineering. However, even if companies producingsuch crystals are promoting their practicality and versatility, inthelastyearsthescientificcommunityisquestioningabouttheirreal utility. As Chalker-Scott fromWashington State Universitypointedoutinherpublicationsonthetopic,sincethecommonlyusedwateringcrystalsaremadeoutofnon-renewablematerials,whosemonomers can be toxic (e.g. acrylamide), the potentialrisks of their usage areway higher than the benefits ofwaterstorageandcontrolledreleasethatcan,inaddition,beobtainedinmanyotherwayswithlowerenvironmentalimpact[41].

Perfume delivery: Duringtheninetiespatentsdescribingvolatilespecies delivery technologies started to grow in number. Inparticular, themost significant patented inventions in the fieldseemtobeissuedbyProcter&Gamble,processingthefragrancesintocyclodextrincomplexes[42-44].

Thegeneralaimwastodevelopdevicescapableofslowlydispensefragrancestothesurroundingsinthelong-termandreplacetheclassic salt-based (sodium dodecylbenzenesulphonate) tabletswith new, more practical and, let’s say it, fancier house caresolutions.Theroleofhydrogelsintheprocessrevolvesaround,once again, their swelling properties that can be exploited inmaterials “wherein release of a perfume smell is triggered bydynamic swelling force of the polymer when the polymer iswetted” [43]. Thesedevices release volatileparticles thanks toosmoticdiffusionofthespeciefromtheswollenhydrogeltonewwaterintheenvironment.

Cosmetics: Cosmetic industry is amarket constantly increasingin dimension and product offer. One of the reasons of thisbehavior is related to the long path to approval necessary formedicaldevices,medicalprocedures,drugsorbiomolecules, inordertomakeallowedtobesoldonthemarket.So,whileontheotherhand,thecosmeticmarketrequires lesstimeandmoneyexpensesfortheapprovalofaproduct,itiscommontotestnewinventions and devices in cosmetics before and in more riskyclinicalapplications.

Foraproducttobeapprovedincosmetics,themostimportantparameter to be assessed is Primary Irritation Index (PII). Thisindex is simple to obtained and exist both for skin and eyes,indeed for each level of PII corresponds a determinate effect[45,46]. Considering that the majority of hydrogels used inthisfieldaresuitable forcellscultureand forotherbiomedicalapplications,isnotsurprisingthattheirIrritationIndexisamongthelowest.Thus,witharelativelysmall investment,companies

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areabletolaunchonthemarketnewcosmeticproductsbasedonhydrogels,suchassocalled“beautymasks”.

Usually made with engineered collagen (MasqueologyTM by SEPHORA USA Inc., BioCollagen Cosmeceuticals byNOVOSTRATAUK Ltd.), hyaluronic acid (SEPHORAUSA Inc.), orpolyvinylpyrrolidone (Pecogel®), these masks claim to hydratethe skin, restore its elasticity and promote anti-aging actions[47]. Pecogel by Phoenix Chemicals Inc., is awide selection ofhydrogels, based on polyvinylpyrrolidone, with differences incomposition and/or crosslinkingmethod. Pecogels are suitableforcosmeticpurposes,suchassunscreencreamormascara[48].Furthermore,insomeofthecommerciallyavailablecompoundssuchasHydroGelFaceMasksbyFruit&PassionBoutiquesInc.,themoisturizingactionoftheseorganicpolymericgelsiscoupledwithmorecomplexdrug-deliverysystemsdevelopedtoreleaseofbiomoleculeslikevitaminCorB3.

The cosmetic industry is on the cutting edge of hydrogels,indeed a pH-Sensitive material P(MAA-co-EGMA) has beendeveloped for release of cosmetics drugs like arbutin,adenosine,andniacinamide,wellknowingmoleculesforwrinkletreatmentandforskin-whitening[49].Thishydrogelschangeispermeability responding to thepH changes:At pH4.0 it holdsthe pharmaceuticals inside the matrix, when in contact withskin, at pH 6 and above, the permeability rise and the drugswilldelivered.Theauthorssaidthatthisbehaviorisduebytheionization/deionizationofMAAcarboxylicgroups.

Plastic SurgeryFrom their first development into the scientific research field,hydrogelwhereseenasgoodmaterialsforapplicationincontactwith thehumanbodybecauseof their extracellularmatrix-like(ECM-like)properties[7,50].Thisisthemainreasonwhyattemptsweremadetointroducehydrogelslikenewmaterialsforplasticreconstruction.

Onthispath,formanyyears,HyaluronicAcid(HA)wasthoughtlikethepanaceaforeverypain[51].Isnotsurprising,consideredthis, that HA has been studied to be applied in tissue fillingapplications. One notable company operating in the field isMacrolaneTM.

Starting in 2008, Macrolane’s treatments and products werespecifically studied toenhancebreast sizeandshapeandoffera more biocompatible alternative to standard and aggressivesilicone prosthesis. Anyhow, soon the scientific communitystarted to point out the controversial behavior of theseprocedures in latter mammographies: briefly, HA worked as ashielding agent, appearing as denser tissue, and consequentlyruining theoutcomeof theexam.ThusnowadaysMacrolaneTM isusedfordiverselysituatedfillingwiththeexceptionofbreasts.Thecompoundisinjectedinsidethebodywithasyringeandletit gels restoring the volume. This procedure started to receivecriticisms by the scientific community, but yet at themomentthereisstillnotasufficientamountofpublicationsinvestigatingthe properties and possible risks of the gel, concerns startedto rise in particular about the long term side effects of thistreatmentandmorespecificallyaboutHA’sroleincancercontrolanddevelopment[52,53].

Anotherpromisinguseofhydrogelsisbulkingagentsfortreatmentofurinaryincontinence:smartinjectablegelscanbeinvolvedinclinicalprocedureswherethesematerialscanbeusedtotightenthe urethral channel and reduce patient’s incontinence. Withsuchasimplesolution it ispossible toeraseorat least reduceaconsistentsocialhandicapandhelppatientstoholdanormallife[54].AnexampleofproductusedinthisfieldisBulkamid®byConturaInternational,apolyacrylamidehydrogelisacommercialproductdevelopedtohelpwomenwithincontinencenarrowingthe conduct. There are few motive for the incontinence,and an exhaustive classification is made by Lose et al. wherethey subdivide each class, Bladder / Urethra incontinent, inoveractivity / underactivity, in theirwork theywrote that bulkinjectablesystemsarepromisingtoimproveurethralcoaptation.Problematics like infections and body response have beenevaluated. In a sample composed by 130 women researchershavefound10patientswithinfections,5withinjectionsitepain,2withincontinenceandonly1withinjectionsitelaceration.

Immunotherapy and vaccineAnanovectorcomposedofpeptide-basednanofibroushydrogelcancondenseDNAtoresultinstrongimmuneresponsesagainstHIV. HIV infection is presently incurable and has led to morethan30milliondeaths[55].Traditional,vaccinessuchasliveorattenuatedvirusesare ineffectiveandposepotential risks [56].Asaconsequence,safealternativessuchasDNAvaccineshaveattracted great attention, they are easily degraded by DNasesand lysosomes, and injected naked DNA plasmids are poorlydistributed and inefficiently expressed; thus,DNAvaccines canonly induce modest humoral and cellular immune reponses[57]. As such, DNA vaccines must be injected with a deliverysystem to enhance the immune responses. Different syntheticdelivery systems have been constructed including polymers[58], liposomes [59] and nano- or microparticles [60]. Somedisadvantages limit their further usage, such as toxicity, smallamount of antigen loading anddecreasedbiological activity ofDNAduringcomplexpreparation.

Nanofibersprovideanidealplatform[61].Anotherplatformsuitedfor DNAdeliveryisbasedonhydrogelbecauseofitshighloadingcapacity, mild working conditions, and good biocompatibility[62]. Supramolecular hydrogel is a kind of hydrogel composedof nanofibers formed by the self-assembly of small molecules(molecular weight usually <2000) in aqueous solutions [63].They can quickly respond to various external stimuli as: pH,temperature,ionicconcentrationoradditionofenzymes.

Recently, peptide-based delivery system emerged as analternativemeanstoefficientlyintroducegenesordrugsintocellsboth in-vitroand in-vivo [64]. Someenzyme-triggeredpeptide-based nanofibrous hydrogels are useful for the entrapment ofdrugmoleculeswithoutreducingtheiractivitybecausethattheycanworkinmildconditions[65].Theyarealsobiocompatibleandhavewell-definedstructures.

Nap-GFFY (Naphthalene acetic acid-Glycine phenylalaninetyrosine) was demonstrated as a short peptide to constructgelators of nanofibrous hydrogel. This nanovector can stronglyactivate both humoral and cellular immune responses to a

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balancedlevelrarelyreported,whichiscrucialforHIVpreventionand therapy. In addition, this hydrogel shows good biosafetyin-vitro and in-vivo. Such hydrogel have a critical nanofibrousstructure for the dramatically improved immune responsescomparedtoexistingmaterials.Thereasonofthishighefficiencyis that this nanovector can condense DNA, promote DNAtransfection,andenhancegeneexpressionin-vitro.Furthermore,it shows promising biocompatibility and no obvious toxicity.This peptide-based nanofibroushydrogel used as a HIV DNAnanovectorcanopenthedoorsforeffectivevaccinationbasedonpeptide-basednanofibroushydrogels[66].

Environmental applicationsOver past the years, nations gradually started to care aboutenvironmentalissuesandpollution.Manygovernmentsdecidedtooptforgreenerandsaferfortheenvironmentpolicies.Waterpollution is one of the biggest issues afflicting especially poorareasofAfrica,AsiaandSouthAmerica.Thankstotheiraffinityforwater,hydrogelsmightbeusedintwodifferentwaystotreatwatersource.

First the matrix can be used as a holder for purifyingmicroorganism.Manyinterestingstudies,onthisparticularpath,weredevelopedbyencapsulatingmicroorganismsinsidediversecarrier materials [67]. Chlorella and Spirulina are the mostused ones. Thesemicroorganisms are already used to removepollutantschemicals fromwater resources.The idea is tokeepthe bacteria inside the network and consequently protect andcontrolthebacteria-colturewhilecleaningthesiteofdepuration.Bothsyntheticandnaturalhydrogelswerebeenused.ThebestworkinghydrogelsinliteratureappeartobeAlginatederived[67]oralternativelycarrageenanandagar[68].

Asecondinterestingwaytosolvetheproblemofpollutantsistomodify thehydrogels to let them seize and keep thepollutantinsidethenetworks.Manyauthorshavetriedthiswaytoseizemetalions:thegroupofIranihassenttopublishapaperinwhichtheydiscussanewcompositehydrogelforPbI(II)removal.Briefly,they created a polyethylene-g-poly (acrylic acid)-co-starch/OMMT (LLDPE-g-PAA-co-starch/OMMT) hydrogel composite,usingitlikeanadsorbentpollutant(Pb(II))remover.TheyputthehydrogelinasolutioncontainingleadacetateandthenmeasuredtheadsorptionwithanAtomicAbsorptionSpectrometer (AAS);afterthattheydidade-absorptionphaseandrepeatforseveralcycles.Theelectrostaticattractions,ionexchangeandchelationare possible explanation for the metal adsorption happenedduring the experiments. They reported that the equilibriumadsorption data of the hydrogelwas consistentwith Langmuirisothermandthe430mg/gadsorptioncapacitywasinlinewithothercommonadsorbents[69,70].

Anotherfeasiblewaytoachieveaninterestingwaterfilteringisexplained inapaperbyYanetal.,wherethegroupperformedetherification and consequent functionalization of chitosanbeads in order to obtain carboxymethilated chitosan with anenhanced adsorption of metal ions. This has been proven toimproveselectiveadsorptionofspecificionslikeCu(II),Pu(II)andMg(II) [70]. It is interestingproperties that canbeexploited in

dye removal application and that has been demonstrate to bepossible by magnetical doping of hydrogel microspheres withinterpenetratednetwork(IPN)structures[71].

Hydrogels could be either used like a probe to detect heavymetalionslikeintheworkbyWangetal.[72].Moreover,otherapplicationofpolyacrylamidegelsisafloodcontroldevicecalledWATERGELBAG®andproducedbyTaiHeiCo.,Ltd.

One of the biggest environmental problems currently hardto solve is for sure the loss of oil in seas and in other watersources.Inthepastyearsthequantityofoilsubstancedispersedinto the hydrosphere around the world is risen dramatically.Food processing, hydrocarbons industry, refining process haveincreased the riskofpollution. Itwasdiscovered thateffluentsreceiving wastewaters from industry have 40.000 mg/L oilconcentration[73].

Different kind of depolluting system have been attempted likebentonite organically [74,75], palygorskite [76], a magnesiumaluminumphyllosilicate,andactivatedcarbon. Inastudydated2010authors attempted todevelopahydrogel to retainwaterwith oil-pollutantmolecules [77]. They claim a very promisinghydrogelischitosanthankstoahighpresenceofaminogroupsand hydrogen that can react with vinyl monomers. Authorsexplaineduseofpolyacrylamidegraftedonthepolysaccharidesrisesthecapabilitytoattractandholdsolidparticleswhichremaininsuspensioninwater,calledflocculants.Thehighercrosslinkingdegree,thelowertheretainingcapability.Thisisduetoasmallerdistance between two nodes in thematrix network. Reducingthisdistance,theswellingdegreewilldecrease.Furthermoretheinitialconcentrationofwastedoilintheenvironmentisanotherfundamentalparameterbecauseofitspropertyofaffectingtheadsorptionkinetic.Infactahigherconcentrationcorrespondstofasterswellingkinetics.

Bacterial cultureAs already discuss for the environmental applications section,hydrogels can hold inside their matrix a significant numberof microorganism for purification of water, for production ofbiomolecules,orforsimplecultureofbacteriabythemselves.

Indeed, agar is famous as the golden standard substrate forbacterialcultureinbiotechnologicalapplications[78].Sinceitisindigestibleby a great numberof bacteria andmicroorganism,it provides a perfect environment for their culture on a solidsubstrate [79]. Different kinds of agar are been studied, eachwith a potential use for likewise different kinds of bacterial.Between thembrucella agar, columbi agar, schaedler agar, or trypicasesoy agar are the most common. None of these gelshassuperiorresultscomparedtotheothers,insteadeveryoneissuitablefordifferentapplicationsinreasonoftheirdifferentprosandtheircons[80,81].

Electrophoresis and proteomicGel electrophoresis currently represents one of the moststandard techniques for protein separation. In addition to themostcommonlyemployedpolyacrylamidecrosslinkedhydrogels,

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acrylamideagarosecopolymershavebeenproposedaspromisingsystems for separation matrices in two-dimensional (2-D)electrophoresis,becauseofthegoodresolutionofbothhighandlowmolecularmass proteinsmade possible by careful controlandoptimizationofthehydrogelporestructure.Asamatteroffact,athoroughunderstandingofthenatureofthehydrogelporestructureaswellasoftheparametersbywhichitisinfluencediscrucial for thedesignofhydrogel systemswithoptimal sievingproperties.

What is theproteomics?The termproteomicswasfirst coinedin1995[82],Proteomicsisthestudyoftheproteome,andisthelarge-scale study of proteins, particularly their structures andfunctions, in a given type of cell or organism, at a given time,under defined conditions, tomake an analogy with genomics,the study of the genome. The proteome is the entire set ofproteins,producedormodifiedbyanorganismorsystem.Thisvaries with time and distinct requirements, or stresses, that acell or organism undergoes. Proteomics is an interdisciplinarydomainformedonthebasisoftheresearchanddevelopmentoftheHumanGenomeProject.Whileproteomicsgenerally refersto the large-scale experimental analysis of proteins, it is oftenspecificallyusedforproteinpurificationandmassspectrometry.

In thefieldofproteomics, theability todetecta largenumberofproteinsinasingleanalysisrepresentsakeyissuetoachievefast andefficientoperation [83]. In this context, the combineduseof2-Dgelelectrophoresiscoupledwithmassspectrometryhas allowed enormous advances during last few decades andhasbecomenowadaysoneofstandardapproachesforproteinsseparationandidentification(Figure 3) [84,85].

Among the materials used for 2-D gel electrophoresis,polyacrylamide crosslinked hydrogels have been extensivelyinvestigated in the literature [86], because their tunablemeshsize porosity appears to be ideal for separating proteins andDNA samples. Typically acrylamide concentrations higher than5%areusedtoformtheseparationmatrix,withtheacrylamideconcentrationbeingselectedtomaximizeresolutionoftherangeof proteins of interest. Lower acrylamide concentrations arenecessarywhenresolutionoflargehighmolecularmass(HMM)proteins (>500kDa) is sought, however at the expense of poormechanicalstabilityofthegelmatrixthatoftenyieldsdifficultiesinhandlingthesemedia[74].Otherpolymericsystemsalternativeto polyacrylamide gels have also been proposed as separationmatrices for 2-D electrophoresis including agarose, modifiedpolyacrylamidegelsandacrylamide-agarosecopolymers[87-89].In particular, the advantagesof the acrylamide-agarose systemmainlylayinthepossibilityofimprovingtheresolutionoflargeHMM proteins without compromising the resolution of lowmolecularmassproteins,partlyduetotheoptimalaverageporesizeofthesematerials.

Indeed, the electrophoresis migration process through thepolymeric gelmatrix is drivenby the interactionsbetween theproteinfragmentsandtheporousnetworkofthegel,causingthequalityofproteinresolutiontobehighlydependentondifferentstructuralparameterscharacteristicofthegelmatrix[90].Amongthese,meangelporesize,poresizedistributionandstiffnessof

thegelplayacrucialrole.Inordertoachieveimprovedseparationperformance in2-Delectrophoresisapplications, it is thereforeessentialtounderstandthespecificnatureoftheporestructureofthegelandtheparametersthroughwhichthisporestructurecanbecontrolledandmanipulated[91,92].Inparticular,itisofgreat interest to investigate structure-property relation-shipsof these hydrogels in the attempt to optimize their functionalperformance.

A wide large range of hydrogel chemical compositions wasstudiedandtheireffectonstructuralandfunctionalpropertiesofthehydrogelwaselucidated.Byemployingdynamicrheologicaltestsandcreep-recoverytests,acorrelationwasfoundbetweenthe rheological response of these hydrogels and their sievingproperties.

More specifically, the evaluation of the crosslinking density bymeansof dynamic tests and theuseof viscoelasticmodels fordetermining the resistance of non-permanent crosslinks tomove in thenetwork systems shed light on thepore structureofthehydrogelmatrixandhelpedtoclarifyitsinfluenceontheelectrophoreticseparationperformance.Themechanicalstabilityof thecrosslinkedhydrogelswasalso investigatedbymeansoftensiletestsandcorrelatedwiththecrosslinkingdensityofthegelmatrix.

Interpenetrating Polymer Networks (IPN)Interpenetrating polymer networks (IPNs) hydrogels havegained great attention in the last decades,mainly due to theirbiomedicalapplications.IPNsarealloysofcrosslinkedpolymers,atleastoneofthembeingsynthetizedand/orcross-linkedwithintheimmediatepresenceoftheother,withoutanycovalentbondsbetween them, which cannot be separated unless chemicalbongs arebroken [93]. The combinationof thepolymersmusteffectively produce an advanced multicomponent polymericsystem,with a newprofile [94]. According to the chemistry ofpreparation,IPNhydrogelscanbeclassifiedin:

i- SimultaneousIPN:Whentheprecursorsofbothnetworksaremixed and the twonetworks are synthesized at thesametimebyindependent,non-interferingroutssuchaschainandstepwisepolymerization[95].

ii- SequentialIPN:Typicallyperformedbyswellingofasingle-networkhydrogel intoa solutioncontaining themixtureof monomer, initiator and activator, with or without acrosslinker.Ifacrosslinkerispresent,fullyIPNresult,whilein the absence of a crosslinker, a network having linearpolymers embedded within the first network is formed(semi-IPN)[96].

When a linear polymer, either synthetic or biopolymer, isentrapped inamatrix, forming thusa semi-IPNhydrogel, fully-IPNcanbepreparedafterthatbyaselectivecrosslinkingofthelinearpolymerchains[97,98].

Evenifitisstillachallengingtask,thesynthesisofionimprintedIPNhydrogelsconstitutesapromisingdirectioninincreasingtheselectivityof thisnovel typesofsorbents,which isexpectedtoreceivemuchattentioninthefuture.

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Tissue Engineering (TE) ApplicationsHydrogelsarethreedimensionalpolymerscaffoldsusedinseveralapplicationsoftissueengineering.Aparticularlyimportantgroupoftechniquesisthesocalledin-vivotissueregeneration.Inthiscase,apatient’sowncellsarecombinedwiththepolymer,andheld in-vitro until ready to be implanted. The hydrogel acts asa natural extra-cellularmatrix that subsequently promotes cellproliferation and tissue re-growth. The pseudo-extra-cellularmatrix, comprised of growth factors, metabolites and othermaterials,bringscellstogetherandcontrolstissuestructurewiththeultimategoalofreplacingthenaturaltissuethatwaslostordamaged.

Whenpartsofthewholeofcertaintissuesororgansfail,thereareseveraloptionsfortreatment,includingrepair,replacementwith a synthetic or natural substitute, or regeneration. Thefigurebelowshowshowtissueororganinjury,diseaseorfailurehas evolved to reach the field of tissue engineering. Tissuerepair or replacement with a synthetic substitute is limited tothose situations where surgical methods and implants haveachieved success. Although implants have been a reasonablysuccessfuloption,tissueengineeringholdsoutgreatpromiseforregenerationofthefailedtissue.Thefirstoptionofthediseasedorinjuredorgansisextracorporealtreatment,inwhichbloodiscirculatedthroughpolymericmembraneexchangedevices.Thesedevicesareusuallypassiveexchangesystems,butmorerecentlyexperimental systems may contain entrapped or encapsulatedcellsfromotherhumanoranimalsources.Thoselattersystemsare called bio-artificial or bio-hybrid organs. Total replacementofthediseasedormalfunctioningorganortissuewithanaturalsubstitute requires transplantation of an acceptable, healthysubstitute,andthereisalimitedsupplyofsuchorgansandtissues.Thus,tissueengineeringholdsoutgreatpromiseforregeneration

oforgans(Figure 4). Hydrogelshavebecomeincreasinglystudiedasmatricesfortissueengineering[85].

Hydrogelsdesigned foruseastissueengineeringscaffoldsmaycontainporeslargeenoughtoaccommodatelivingcells,ortheymaybedesignedtodissolveordegradeaway,releasinggrowthfactorsandcreatingporesintowhichlivingcellsmaypenetrateand proliferate. Table 1 lists parameters and properties ofhydrogelsfortheseapplications.

One significant advantage of hydrogels as tissue engineeringmatricesvs.morehydrophobicalternativessuchasPLGAistheeasewithwhichonemaycovalentlyincorporatecellmembranereceptor peptide ligands, in order to stimulate adhesion,spreading and growth of cells within the hydrogel matrix.However, a significant disadvantage of hydrogels is their lowmechanical strength, posing significant difficulties in handling[99].Sterilizationissuesarealsoverychallenging.Itisclearthattherearebothsignificantadvantagesanddisadvantages to theuseofhydrogelsintissueengineering,andthelatterwillneedtobeovercomebeforehydrogelswillbecomepracticalandusefulinthisexcitingfield(Table 2).

Itshouldbenotedthatagelusedasatissueengineeringmatrixmayneverbedried,butthetotalwaterinthegelisstillcomprisedofboundandfreewater.

Langer has described tissue engineering as an interdisciplinaryfield that applies the principles of engineering and the life

Transplantation

(if available)

Synthetic implant

Implantation of scaffold + cells

TE Treatment

Culture of cells on polymer substrate

(scaffold)

FORMATION OF NEW AUTOLOGOUS TISSUE

Tissue/Organinjury,

Disease or failure

RestoredFunction

---------

Immunogenity Issues

RestoredFunction

---------

BiocompatibilityIssues

Figure 4 Evolution of various therapeutic methods for treatinginjured or diseased tissues and organs, to tissueengineeringfortherepair,regenerationorreplacementofsuchtissuesororgans.

Type of Hydrogel

Molecular structures

Composition of Hydrogel

Important properties

• Physical• Chemical

• Linearpolymers

• Blockcopolymers

• Graftcopolymers

• Interpenetratingnetworks(IPNs)

• Polyblends

• Naturalpolymersandtheirderivatives

• Syntheticpolymers

• Combinationsofnaturalandsyntheticpolymers

• Degradability

• Injectability

• Mechanicalstrength

• Easeofhandling

• Shapeandsurface/volumeratio(sheets,cylinders,spheres)

• Closed/openpores

• Watercontentandcharacter

• Chemicalmodification(e.g.havingattachedcelladhesionligands)

• Additionofcellsand/ordrugs

• Sterilizability

Table 1 Highlight on important properties and characteristics ofhydrogelsfortissueengineering.

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sciences to the development of biological substitutes thatrestore, maintain, or improve tissue function [100]. This fieldis currently advancing rapidly, combining progress in biologyand technology,andhas raisedmanyhopes inseveralareasofbiologyandmedicine.Firstandforemostithasastrongpotentialas an application in regenerativemedicine, for developing life-likereplacementtissuesandorgans.Butitalsohasanimportantrole to play in fundamental biological research; it allows usersto reproducephysiologicalmicro-environmentsmore closely inin-vitrosettingsthantraditionalculturemethods,offeringawaytobridgethegapbetweenin-vivoexperimentsandconventionalin-vitro studies. On amore operational side, the developmentof new in-vitro models based on human cells has raises thepossibilityoftacklingmanyoftheobstaclesthatcurrentlyhinderpharmaceutical research and drug development. Engineeredtissuescouldreducethelimitationsrelatedtothetranspositionoffindingsfromoneorganismtoanother,alleviateethicalproblemsrelated to animal testing, increase standardization, and allowmore thorough studiesof toxicity,metabolismand life cycleofputativedrugsbeforeenteringclinicaltesting,thiscould,inturn,reducetheduration,costfailurerateandriskofclinicaltrials.

In vivo, the formation of organs and tissues is based on thecoordination in time and space of cell differentiation, polarity,shape,divisionanddeath.Thiscoordinationreliesonthecellularintegrationofsignalsfrommicroenvironment,mainlyconsistingoftheextracellularmatrix(ECM),andintercellularcommunication[101].Thetransductionofatypicalcombinationofthesefactorscoupledtospecificcytoplasmiccomponentscaninducethethreeprogressivestepsindifferentiation.First,stemcellsarespecifiedtowardsacertainfate,thentheyshiftfromaspecifiedstatetoadeterminedstate,inwhichthecellfatecannotbereversed,andfinallyreachtheirdifferentiatedstate.

Themainchallengeoftissueengineeringistoreconstitutein-vitro anenvironmentthatinducesthedifferentiationofcellsandtheirorganization in anordered functional tissue. The cell substrateisofparticular importance,since in-vivo theextracellularspaceisoccupiedby theECM.Thechemicalcompositionof theECMand the resulting mechanical properties are both importantaspects,asthetransductionofbothchemicalandphysicalsignalsvia cellular adhesionmolecules affects cell shape, polarization,migration and differentiation [102]. Furthermore, the ECM

topographyorientstissuepolarityandthemorphogenesisofneworgans.

Molecularhydrogelsholdbigpotentialforcellscultureandtissueengineering;peptide-basedmolecularhydrogels,especiallythoseof long peptides, could provide suitable environments for cellgrowth,divisionanddifferentiation.ZhanggroupandtheStuppgroup have demonstrated that peptide-based hydrogels couldguidethedifferentiationofstemcells[103].Thechallengeinthisfieldwasamethodtoseparatecellsfromgelspost-culturesuchas using short-peptide-based molecular hydrogels formed bybiocompatiblemethodsfor3Dcellculture,stemcellcontrolleddifferentiation and cell delivery [104]. Responsive molecularhydrogelsfortherecoveryofcellspost-culturewerealsostudied[105].

Collagen is the most abundant protein mammals, making upabout30%oftheoverallbodyproteincontent.Inordertomimiccollagennanofibers,a serialof shortpeptidesbearingcollagenrepeatingtripeptideofGly-Xaa-4-Hyp(GXO,XwasLys(K),Glu(E),Ser(S),Ala(A),orPro(P))wassynthesized[106].

Therearetwomainbottlenecksrightnowintissueengineering.Thefirstoneiscorrelatedwiththepossibilityofobtaining,in-vitro, developedvasculartissue[107,108].Insecondinstance,onceapseudo-physiologicalangiogenesisisachieved,theambitiousaimistocreateengineeredwholeorgans[109].Firstattemptsinthisdirectionwheremadebyprinting3Dorgan-likestructureswithECMandcells,orbydecellularizationandseeding.

Under these circumstances, hydrogels are usually studied astemporarysubstitutesoftheextracellularmatrix(ECM)becauseof comparable physico-chemical properties, such as stiffnessand hydrophilicity [51]. Hydrogels used as scaffold in TE mustbebiocompatibleandelicit thesmallest responseby thebody.This is a very complicated problem, indeed, for the culture ofeukaryotic cells implies a lot of precautions. Cellsmorphology,metabolismandoverall phenotypearedirectly correlatedwiththesignalstheyreceive,firstofallfromthephysicalandchemicalspropertiesofsubstrate.

Themicroandnanostructure,theporosityandthestiffnessofthesurfacesareall important signals for thecells (topographyandmechanicalproperties)[109].Averyimportantphenomenon,forinstance, is thecontact guidance, introducedbyWeiss in1934[110],forwhichcellsalignstotheshapeandthemicrostructureofagivensurface.Especially forsomekindofcell culture (e.g.myocytes), this phenomena is crucial for the creation of newworking tissue [111,112]. Hydrogels for TEmust be developedattemptingtofitthisnecessity.

For the purpose, different materials have been investigated.Actually research is focused mainly on degradable scaffold toallow cell migration while thematrix degenerates. Researchesdemonstrated the importance of engineered degradation onnon-degradable scaffolds by viability test [113,114]. Thesehydrogelscanbedividedbytheoriginof thepolymertheyaremadeof:eitherifitisnaturalorsynthetic.Inthepresentarticletheattentionwillbefocusedonnaturalderivedhydrogels

Dextran: Dextran is natural polysaccharide obtained from the

Advantages Disadvantages• Aqueousenvironmentcanprotectcellsandfragiledrugs(peptides,proteins,oligonucleotides,DNA)

• Goodtransportofnutrienttocellsandproductsfromcells

• Maybeeasilymodifiedwithcelladhesionligands

• Canbeinjectedin-vivo asaliquidthatgelsatbodytemperature

• Usuallybiocompatible

• Canbehardtohandle• Usuallymechanicallyweak• May be difficult to load drugs andcells and then crosslink in-vitro as aprefabricatedmatrix

• Maybedifficulttosterilize

Table 2 Importantadvantagesanddisadvantagesofhydrogelsasmatricesfortissueengineering.

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digestionofamylopectin.Lowmolecularweightdextranisbeenusedlikeaplasmaexpanderthankstotherelativelyinertandnon-toxicbehaviorofthispolysaccharide[115].Themostinterestingcharacteristic of dextranareits protein rejection properties, theso called non-fouling [99], coupledwith great biocompatibilitydue to its glycocalyx mimic behavior [100]. This property isuseful to create an ECM-like hydrogels for tissue engineering,for instance, Yunxiao and collaborators created a copolymerbetweenmethacrylate-aldehyde-bifunctionalized dextran (DEX-MA-AD)andgelatinB.Thismaterialwasobtainedbyultraviolet(UV)-crosslinkingbetweenamethacrylategroupsonDex-MA-ADandthealdehydegroups,allowingtheinclusionofgelatininthematrixthatgrantingenzymaticallydegradationandcelladhesiveproperties.Researchersdemonstratethatthiskindofhydrogelscouldpromoteadhesionofvascularendothelialcells[116,117].

Gelatin: Gelatin is the denatured form of collagen, one ofthe major component of ECM. Collagen however carriesimmunogenicityproblemsduetothepresenceofantigensfromtheoriginal tissue.Gelatin is a proteinmaterialswith a longαhelixwith a high content in glycine (≈25%) [118].Gelatin existintwodifferentform,processedinacidsolution(typeA,usuallyporcine)orinalkalisolution(typeB,usuallybovine)[119].Initsnaturalformisquicklydissolvedinwater,butitcanbecrosslinkedto obtain a hydrogel with higher mechanical properties anddegradationrate.

Thecrosslinkingcanbeperformedinmanydifferentways,fromthe physical side we could use UV reticulation or the chainpolarity.Fromthechemicaloneisverycommontopolymerize,withenzymestoo, thechainswithaboundonthesidegroupsofaminoacids.LysineandGlutamicAcidarethemostemployedforit.

Thanks to his similarity to the natural ECM,many compoundswerebeendevelopedwithgelatin-coatingorincludedelement.Chitosan-gelatin, fibroin-gelatin, alginate-gelatin, dextran-gelatin are very common. Das and collaborators attempted tocreateafibroin-gelatinbiomaterialforbioprintingcells-ladenina3Dtissueconstructs.Theydevelopedtwokindsofhydrogels.One crosslinked by sonication, the other crosslinked usingtyrosinaseenzyme.The results showed that sonication-gelatin-fibroin hydrogel shown better osteogenic differentiation whiletyrosinased-gelatin-fibroin supported better chondrogenic andadipogenicdifferentiation[120].

Chitosan: Chitosan is a polysaccharides from chitin of thecrustacean skeleton. It is composed by the repetition ofN-glucosamine units. A crucial index to assess chitosan’sproperties is thedegreeof acetylation,definedas thenumberofamineinthematerial.Forinstance,itisprovedthatchitosancould decrease the adsorption of protein and the binding ofbacteria [121]. Actually it is yet not clear how this materialscouldbeenattachedbycells.Somestudiesreportafirstperiodinwhichchitosanrepelledcelladhesion,andasecondinwhichcellsstartstobindtoit.Thereisnoauniformityinresultsaboutthisrefractory-period, anyhow it canbeexploited to seeddifferentkindof cells. For example, Tao Janget al. published a study inwhich they used photo polymerizationon chitosan in order todeposit cellsbetween thechitosanpatterns.They suggest that

after the refractory-period, while cells are disposing in no-chitosancoatedregions, it ispossible toseedsanother typeofcellsforthedevelopmentofamorecomplexsystem[122].

Inanotherstudychitosanhasbeencoupledwithgelatintocreateanin-situgelforcellseedingand/ordrugdelivery. Inparticularthegroupevaluatedthedifferenceincrosslinkingbytwodifferentenzyme[123].

Hyaluronic acid: Hyaluronic Acid – HA is a GlycosaminoglycanGAG enclosed in the natural ECM, core of the material is apolysaccharidewithhighaffinity forwater.Usually, to increasethemechanicalpropertiesofthisbiomaterial,acovalentcross-linkingbetweenchainsisdone.Tezel&Fredricksonhavereportedthat a too high degree ofmodification and cross-linking couldinfluencethebiocompatibilitypropertyofthematerial[124].

In vivo HA can have different molecular weights. Low andhigh molecular weight HA cause an opposite cells behavior[125]. HA macromolecules shown an anti-inflammatory,immunosuppressive properties and blocks angiogenesis, whilecleavedsmallfragmentsinducetheoppositebehavior,enablingendothelialcellsmigrationandangiogenesis [126,127]. Indeed,low molecular weight HA have been correlated with somecancers,likeprostaticone[125,128].

HAhydrogelswereobtained inorder toexploit theangiogenicpower of the molecule during the degradation of a material.Kisiel’s group, for example, crosslinked HA with protease-degradablepeptidesandaddedcelladhesionligandstoimprovethecellspreadingonthematerial[129].

Onanotherhand,ShuetAl.triedtomimictheECMcopolymerizingHAandgelatin.TheyaddedathiolgrouptotheHyaluronan, inthiswaytheywereabletocrosslinkedmodified-Hyaluronanwithmodified-Gelatinbydisulfidebonds[130].

Pectin: Another polysaccharides used in tissue engineeringhydrogels is pectin. It is obtained from cells walls after a lowpH, high temperature processing. Unfortunately, until now,researchershavenotreachedthegoaltostandardizethisproductinaneconomicallysustainableway[131].

Based on its esterification degree pectin is classified from lowmethoxyl to high methoxyl. Tuning this property changes themechanicalbehaviorof thematerial.Reticulation canoccurbyloweringpHtoobtainphysicalgel,orusingdivalentortrivalentionstoobtainwater-insolublegel[35,132].

In the tissue engineering field, pectin seems very interestingbecauseof itspromotionofnucleationofmineralphasewhenimmersedinaspecificbiologicalsolution[131,133].

Alginate: Derivedfrombrownalgae,alginateisapolysaccharidecomposed of beta-D-mannuronic acid and alfa-L-gluronic acid.Itsreticulationcanalsooccurbydivalentcations(Ca2+,Fe2+, Ba2+)[134].

Intissueengineeringalginatecanbeusedasanimmunoisolationbarrier [117].Alginate scaffold for the regenerationof annulusfibrosus are also been developed. This hydrogels have shape-memorycapability,arecytocompatibleandsupportsproliferationandmetabolicactivity[135].Moreover,alginatehydrogelswere

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used to reduce liver cellsdeath [136]andwith silk-fibroinasasubstrateforstemcellsculture[137].

Culture of organs-on-chipsRecenttrendstowardsthedevelopmentofin-vitromulticellularsystems with definite architectures, or “organs on chips”are studied. First, the chemical composition and mechanicalproperties of the scaffold have to be consistent with theanatomical environment in-vivo. In this perspective, theflourishing interest in hydrogels as cellular substrates hashighlighted the main parameters directing cell differentiationthatneedtoberecapitulatedinartificialmatrix.Anotherscaffoldrequirementistoactasatemplatetoguidetissuemorphogenesis.Therefore specificmicro-fabrication techniques are required tospatially pattern the environment atmicroscale. 2D patterningisparticularlyefficientfororganizingplanarpolarizedcelltypessuchasendothelialcellsorneurons.However,mostorgansarecharacterizedbyspecificsubunitsorganizedinthreedimensionsat the cellular level. The production of such 3D patterns in-vitro is necessary for cells to fully differentiate, assemble andcoordinatetoformacoherentmicro-tissue.Thesephysiologicalmicrostructures are often integrated in micro-fluidic deviceswhose controlled environments provide the cell culture withmore like-life conditions that traditional cell culture method.Suchsystemshaveawiderangeofapplications,forfundamentalresearch, as tools to acceleratedrugdevelopment and testing,andfinally,forregenerativemedicine.

Thein-vivoextra-cellularmatrix(ECM)consistsmainlyofcollagenfibers, elastin fibers, glycoproteins and polysaccharides. It actsas a mechanical support to the cells it surrounds and playsan important role in cell shape, cell polarity, cell migration,resistance to external forces, and signal transduction. Theprimary focusoftissueengineering is toachievean in-vivo-likecellular environment, by developing in-vitro artificial ECM. Inregenerativemedicine applications, such scaffolds could eitherbedirectly introduced intoan injuredorgan inorder to inducein-vivo genesis, or first seeded with cells in-vitro and thentransplanted once those cells have differentiated. For in-vitro research and testing applications, they are generally directlyusedasasubstrate.Inallcases,theartificialstructuresneedtorecapitulatethe in-vivoenvironmentsignalsresponsibleforcelldifferentiationintothedesiredtissue.

Biomaterial scaffolds are essentially made of hydrogels. Basedontheirabilitytoretainwaterbyswelling,theymimicthehighwater content of the extracellular matrix [138]. Cell adhesionligandsmust be present in hydrogels to allow cells to adhere,spread, migrate and proliferate. There is a large variety ofadhesionmolecules, such as laminin and its derivatives [138],fibronectin [139] and collagen [140]. It is therefore crucial toselect the adhesion molecules for which the seeded cell typehas the largest affinity to make adhesion effective. Naturalhydrogels are bioactive and usually provide native adhesionsites.Conversely,synthetichydrogelsareinert,sincetheircarbonskeletonpresentsnoadhesionmoleculesorendogenousfactorsinducingproliferationandcelldifferentiation.

To enrich their potential as bioactive materials, synthetic

hydrogelsaregenerallysupplementedwithadhesionmolecules[141], either by covalent grafting, adsorption or electrostaticinteraction. Adhesion molecules can be grafted after hydrogelpolymerization, or added to the pre-polymerized mixture andeither physically trapped or chemically incorporated duringpolymerization.Finally, inthecaseofphoto-activatedmaterialssuchasPEGDA,adhesionmoleculescanbechemicallymodifiedto covalently attach to the hydrogel backbone. The grafting ofPEGpolymerhasbeenthoroughlydescribedinthereviewofZhuetal.[142].

Cultureofcellsisverydemandingarea.Itdealswithlivingcellsthat are highly sensitive to their surrounding environment. Italso depends strongly on differentiation, a delicate, complexand still only partly understood phenomenon. It thus requiresthe development of increasingly sophisticated technologies.Although the use of hydrogels as physiological substrates toreplacethein-vivoECMisgloballyaccepted,allparametersmustbe carefullymanipulated to avoid additional unwanted signals.Thesearchforoptimalartificialmatriceshasledtotheinventionofdynamichydrogelsthatcanbetunedtoreproducethein-vivo micro-environment,whichispermanentlyrenewedandcrossedbyvarioussignals.

Despite these major improvements, challenges remain. Untilnow,aphysiologicalsubstratealoneis,inmostcases,insufficientto induce cells to assemble into a multicellular entity andcoordinate their activity to forma functionaltissue.Additionalhydrogel structuring atmicrometer scale is necessary. Inmanycases,auniqueassociationofphysiologicalsubstratecoupledtoa3Dstructurereplicatingthephysicalconstraintsperceivedbythecellsin-vivo willbeneededtoinducein-vitrothedifferentiationofcellsintoafunctionaltissue.

Nonetheless, the vast majority of these ̏organs on chips ̏aremostlystatic,whilein-vivo cellsareoftensubjectedtomechanicaldeformationsassociatedwiththeorgan’sfunction.Furthermore,thebiochemicalenvironmentcanvaryintimedependingonthephysiologyof theentireorganism.Thesephysicalandchemicalstimuligenerallyaffectthedifferentiationandself-organizationofcellsintofunctionaltissuesandarethuscrucialtomimic.Tissueengineering therefore needs to move beyond 3D culture toachieve“4dimensionalstructures“thatencompassthetemporaldimensionoforganfunctionaswellasitspatialones.Combiningthe3Dmicro-structuredtissuesandthedynamicstimuliappearstobethenexttoreachinmicro-organengineering.

Bone regenerationThis work gives a general view on the use of hydrogels asscaffold for bone regeneration, with special emphasis oninjectable systems, membranes for guided bone regeneration,biofunctionalizationandbiomimeticmineralization.

Alginatehydrogelhasbeenshowntobeausefultoolforproducingbone and cartilage tissues [142]. It is very biocompatible inhumans and peptides can also be covalently coupled to themolecules.Alginateisanaturallyoccurringpolymerfoundinkelp(seaweed)thatiscommonlyusedingelformation.

The regeneration of large bone defects caused by trauma

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or disease remains a significant clinical problem. Althoughosteoinductive growth factors such as bone morphogeneticproteinshaveenteredclinics,transplantationorautologousboneremains the gold standard to treat bonedefects. The effectivetreatment of bone defects by protein therapeutics in humansrequires quantities that exceed the physiological doses byseveral orders ofmagnitude. This not only results in very hightreatment costs but also bears considerable risks for adverseside effects. There issues have motivated the development ofbiomaterialstechnologiesallowingtobettercontrolbimoleculardelivery from the solid phase. Importantly, a relatively narrowtherapeuticwindowofgrowthfactordoseisexpectedtoleadtoregeneration,whereastoo lowortoohighgrowthfactordoseswillmost likely lead to severe sideeffects, as shown in animalmodels [143]. Since for the generation of constant growthfactor levels, multiple injections or even continuous infusionof high doseswould be necessary, alternative routes involvingbiologically inspired growth factor delivery are sought. In anideal situation, the localized delivery of growth factors shouldpresent physiologically relevant doses and preserve its activityfor prolonged periods of time. Moreover, carrier materialsshouldfacilitatethecommunicationwithcellsofthehostsoastobeactivelyinvolvedintheprocessofregeneration.Synthetichydrogels, highly swollen three-dimensional (3D) networksof macromolecules, have emerged as powerful candidatebiomaterials to fulfill these requirements [144]. Hydrogels forgrowth factordeliveryapplicationshaveeitherbeengeneratedfromnaturalderivedbiomoleculessuchasalginate,collagen,andfibrinorfromsyntheticpolymersemployingchemicalorphysicalcrosslinkingreactions[145].

Forgrowthfactoradministration,thegrowthfactorcaneitherbefreelyembedded in thehydrogelorbound to it. In the formerapproach growth factor release is driven by passive diffusionor coupled to material degradation. Release kinetics can bevaried by altering material degradation rate or by changinggrowthfactorquantity.Toallowcovalenttetheringtosyntheticor biologically derived hydrogels, GFwere chemicallymodifiedor genetically engineered to contain functional groups such asthiols,acrylates,azides,…AnexampleofgrowthfactordeliveryfromfibrinhydrogelstoregeneratebonetissuewasreportedbyHubbellandco-workers[146].

Recently, a fibrin scaffold possessing integrin-binding sitesadjacent to growth factor-binding sites to obtain synergisticeffectswas used to regenerate bone [147]. For this purpose atrifunctional peptide was created consisting of an N-terminalGlnsequenceforcovalentincorporationintofibrinmatrices,themajorintegrinbindingdomainoffibronectin(FNIII9-10)andattheC-terminusanotherdomainoffibronectin(FNIII12-14)tobindvariousgrowthfactors.SinceFNIII12-14bindsgrowthfactorsfromdifferentfamiliesitallowsforstraightforwarddeliveryofmultiplegrowth factors.BydeliveringBMP-2andPDGF-BB, twogrowthfactorsknowntoinduceboneformationandpreviouslyshowntobindtoFNIII12-14,theauthorstestedforimprovedgrowthfactorefficiencyinectopicpositionsinnudemiceandcalvarialcriticalsizedefects in rats. Byusing growth factor concentrations thatdidn’t show any in-vivo effectswhen delivered in empty fibrin

matrices,theywereabletodemonstratethattheFNIII9-10/12-14functionalizedmatricesenhancedgrowthfactor-inducedboneformationandrecruitmentofboneformingprogenitorcells.Duetotheenhancedgrowthfactorsignaling,concentrationscouldbedramatically reducedandbonetissuedepositionwasobservedwith much lower doses than elsewhere reported, showingpromisefortheuseofsuchimplantsinclinicalapplications.

In another studyanovelphoto-cross-linkable chitosan–lactide-fibrinogen(CLF)hydrogelwasdevelopedandcharacterizedandtheyalsoevaluatetheefficacyofbonemorphogeneticprotein-2(BMP-2)containingaCLFhydrogelforosteogenesis in-vitro and in-vivo.Radiography,microcomputedtomographyandhistologyconfirmed that the BMP-2 containing CLF hydrogels promptedneo-osteogenesis and accelerated healing of the defects in adose-dependentmanner. Thus theCLF hydrogel is a promisingdeliverysystemofgrowthfactorsforboneregeneration[148].

Aphoto-curedhyaluronicacid(HA)hydrogelsweredesignedandprepared containing an osteogenesis-inducting growth factor,GDF-5 (growth and differentiation factors 5). Those hydrogelswerepreparedandconfirmedtohavecontrolledGDF-5releaseprofiles.CytotoxicityandcellviabilitysuggestthatGDF-5loadedHA hydrogel has proper biocompatibility for use as a scaffoldwhich can induce osteogenesis. Moreover, the HA hydrogelshowedimprovedosteogenesisinbothin-vitrotests,theresultsfrom these tests show that the HA hydrogel can be used as ascaffold for bone tissue regeneration [149]. Gelatin hydrogelswerealsoused forbone regenerationatboth the critical-sizedbonedefect.

Cardiac applicationsIn the last decade, advancements have been made towardsdevelopinginjectablehydrogels[179]forthepurposeofcardiacrepair.Hydrogelinjectionsalonehavebeenshowntoattenuatethe decline in cardiac function and left ventricular remodelingtypicallyseenaftermyocardialinfarctioninbothlargeandsmallanimalmodels. Furthermore, hydrogels have also been shownto improve cell retention when co-injected for cellular cardio-myoplastyandtoprolongreleaseoftherapeuticswhenusedasadeliveryvehicle.Thischapterwillreviewthebasicsofhydrogelpropertiesanddiscussrecentstudiesofhydrogelsaloneand inconjunctionwithcellsortherapeuticsforcardiacrepair.

Dental applicationsPulpregenerationtherapyisimportanttoovercomethelimitationsof conventional therapy to induce reparative dentinogenesis.Presently,dentistshavenochoicebuttoremovethewholedentalpulpwithanendodonticprocedurewhenadentindefectwithpulpexposure reachesa critical size resulting inan irreversiblepulp condition. To overcome this limitation, it is consideredimportanttodeveloppulpregenerationtherapyaswellasclarifythemechanismsofpulpwoundhealing.Pulpwoundhealingandregenerationhavecommonprocesses,andresultsofanumberofstudieshaveindicatedthatpulpwoundhealingconsistsofinitialinductions of apoptosis of damaged pulp cells [134],followedby reactionary dentinogenesis by surviving odontoblasts andreparative dentinogenesis by odontoblast-like cells [150,151].Reactionaryor reparativedentin is formed toward the residual

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dentalpulp,however,not in thearea inwhich thedentin-pulpcomplexhasbeenlost.Toachievetheregenerationofthedentin-pulpcomplex,inductionofappropriatepulpwoundhealingandformationofnewdentinindentindefectsareessential,andafewstudieshavereportedvitalpulptherapiestoformnewdentinindefects[152].

Fibroblast growth factor-2 (FGF-2),which is normally stored intheextracellularmatrixandreleasedbyenzymaticdegradationof extracellular matrix molecules, plays a role in physiologicconditions such as enamel and dentin formation of the toothgerm [153], as well as pathologic conditions [154]. It waspreviouslydemonstratedthatagradualandcontinualreleaseofbiologicallyactiveFGF-2wasachievedbyin-vivobiodegradationofgelatinhydrogelsthatincorporatedFGF-2[155].Furthermore,a controlled release of FGF-2 from gelatin hydrogels inducedneovascularizationandregenerationofseveraltissues,includingbone[156],periodontaltissues[157],andothers[158].

Wound healing applicationsWound healing is the promise of a newway to heal damagedskintissuewithhighbiocompatibleandbioactivematerials.Skinburned,diabeticulcer,areproblemsthatatthestateoftheartareveryexpensivetotreat.Prosthetic-tissueengineeredskinarebeenmade, unfortunately they are not ready-to-use; they areexpensive and havemany needs that are not alwaysmatchedby patients. Theoretically, in wound healing applications acrucialparametertoassessisthewoundcontractionthatcanbeevaluated inthisway,rememberingthatA0 is theoriginalburnwoundarea,andAtistheburnwoundareaatthetimeofbiopsy:

[ ] 0

0

100−= ⋅tA AWound Contraction %

AManysystemshasbeenstudied,withorwithoutchemicalstoaidtheskinregeneration.Hyaluronic-acidandgelatinarebothtwopromisingmaterialsfortheaimbecauseoftheirnaturalpresenceinsidehumanECMoftheskintissues.Moreover,inliteraturecanbe found healing systemsmade from cellulose [159], alginate-chitosan copolymers [160], chitosan-gelatin-honey copolymers[161]andnewbiphasicgelatin-silk [162].Mostof theproductsalreadyonthemarketuseacombinationofselectedmaterialsand proper seeding of cells from various origins (allogenic orautogenic), for instance we cite to applications of HYAFFTM esterified hyaluronic acid [163], both produced by FIDIA Ltd.:LaserskinAutograft®,madeofaHA-membranewithkeratinocytes,andHyalo-graft3D®,madewithHA,butwithfibroblastsadded.

Drug delivery applicationsBydrugdeliveryweintendthewholeensembleofprocedures,devices and techniques to avoid the problems of standarddeliveryofmedicals,firstofalltheburstreleaseandquickdecayoftheeffectsofthedrugovertime,especiallyforshorthalf-lifepharmaceuticals[164].

Hydrogels, and smart hydrogels in particular, can be a veryinterestingsolutioninreachingasustainedandtargetedreleaseofpharmaceuticals,bothincreasingtheeffectofthedrugitselfandloweringsideeffectsatthesametime[165].

SilkFibroin,protein-basedbiomaterialwithhighYoungModulus

(1-10GPa)obtainedprocessingthesilkfromvariousinsects,likespideror silkworm (BombyxMori). The silk fiber is composedby fibroin and sericine. This second protein is correlated withinflammatory reactionswhencoupledwithfibroin. Toobtainapurefibroinwire,silkisboiledinalkalinesolutionat120°Cforatleast1hour.Solubilizationandregenerationisnecessaryforusethis biomaterial for tissue engineering, like scaffolding. UsuallythefibroinisprocessedinasolutionofLiBr[166,167],inaternarysolventcomposedbyCalciumChloride/Ethanol/Watertobreakfiber to fiber bond [168] or by SDS [168,169]. This allows thecastingofthepolymerandtheconsequentcreationofdifferentstructureforgeometricalcomplexity.

In2011ChinmoyetAl.usedsilkfibroinfromBombyxMoriandfromNathereaMylitta to createa3D scaffold for cardiactissueengineering. Results showed that metabolic cell response,cardiomyocytes growth and the number of junction betweencellsforAntheraeaMylittafibroinareverysimilartofibronectinones. They concluded that silk fibroin from AM enables anefficientattachmentofcellswithoutaffectingtheirresponsetoextracellularstimuli.

Hydrogels as soft contact lensesContact lenses are one of the reasons why hydrogels wheredeveloped in the first place. They come with many differentfeatures and procedures of usage. In particular, soft contactlenses with a short turnover are the ones for which hydrogelaremostlyuseful.Themostusedpolymerexploitedtoproducesoft-contactlensesissilicone,underhydrogelform,thankstoitspermeabilityforoxygen.

Despitethehighmicrobialkeratitisriskforaprolongedweartime,whichsubsists formostofophthalmicapplications inthisfield,extendedweartimeofsiliconehydrogellensesarefivetimessaferthan other conventional hydrogels forwhat concern infections[170,171].Onlyabout5%ofdrugsadministratedbyeyedropsarebioavailable,andcurrentlyeyedropsaccountformorethan90%ofallophthalmicformulations.Thebioavailabilityofophthalmicdrugscanbeimprovedbyasoftcontactlens-basedophthalmicdrug delivery system. Several polymeric hydrogels have beeninvestigatedforsoftcontactlens-basedophthalmicdrugdeliverysystems:(i)Polymerichydrogelsforconventionalcontactlenstoabsorb and release ophthalmic drugs; (ii) Polymeric hydrogelsforpiggybackcontact lenscombiningwithadrugplateordrugor drug solution; (iii) Surface-modified polymeric hydrogels toimmobilizedrugsonthesurfaceofcontactlenses;(iv)Polymerichydrogelsforinclusionofdrugsinacolloidalstructuredispersedin the lens; (v) Ion ligand-containing polymeric hydrogels; (vi)molecularly imprinted polymeric hydrogels which provide thecontactlenswithahighaffinityandselectivityforagivendrug.

Thesuccessofthetherapyofeyeailmentswithophthalmicdrugsstronglydependsonachievingsufficientdrugconcentrationonthecorneaforasufficientperiodoftime,butthetypicaldeliveryofdrugsbyeyedropswhichcurrentlyaccountformorethan90%of all ophthalmic formulations is very inefficient and in someinstancesleadstoserioussideeffects[172].Uponinstillationinthe eye, thedrugmixeswith thefluid present in the tear filmandhasashortresidencetimeofapproximately2minutesinthe

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film.Onlyabout5%ofthedrugisabsorbedintothecorneaandthe remainingeithergetsabsorbed in theconjunctivaorflowsthroughtheupperandthelowercanaliculiintothelacrimalsac[173].Thedrugcontainingtearfluidiscarriedfromthelacrimalsac into the nasolacrimal duct. The nasolacrimal duct emptiesintothenasalcavity,wherethedrug is thenabsorbed intothebloodstream.Thisabsorption leads todrugwastage,andmoreimportantly, the presence of certain drugs in the bloodstreamleads to undesirable side effects. Furthermore, the applicationofophthalmicdrugsbyeyedropsresults inarapidvariation inthedrugdeliveryratestothecorneaandthislimitstheefficacyofthetherapeuticsystem[174].Inaddition,dosagethrougheyedropsisinconsistentanddifficulttoregulate,asmostofthedrugisreleasedinaninitialburstofconcentration[175].Toincreasethe residence time of the drug in the eye, thereby reducingwastage andminimizing side effects, a number of researchershaveproposedusingcontactlensesforophthalmicdrugdelivery.It has been shown that in the presence of a lens, ophthalmicdrugshaveamuch longer residencetime in thepost-lens tearfilm,comparedwith2minutesinthecaseoftopicalapplicationaseyedrops[176].Thelongerresidencetimewillresultinhigherdrug flux through the cornea and reduce the drug inflow intothe nasolacrimal sac, thus reducing drug absorption into thebloodstream. Several methods have been proposed to makeophthalmicdrugsdeliverablebysoftcontactlens.

The silicone hydrogel lenses can be designed to releaseophthalmicdrugs foranextendedperiodvarying from10daystoafewmonths.ThetransportoftimololandDXinthesiliconegels is diffusion limited, but the release profiles are complexparticularlyfordexamethasonewhichistheevidenceofcomplexmicrostructure of the gels. The silicone hydrogelsmay also besuitable for other drug delivery applications such as punctaplugs,ophthacoils,retinalimplants,transdermalpatches,woundhealingpatches,etc.

Injectable hydrogelsThedesireandneedtominimizetraditionalopensurgeriesandin lightofthechallengesalongwiththetraditional intravenousadministration of chemotherapeutics for example [177],injectable hydrogel-drug system emerges as a powerful toolfor noninvasive and in-situ controlled-release of drugs. Suchapplication could also reduce the healthcare expenses andimprove the recovery time for the patients. Minimal invasiveproceduresusingendoscopes,cathetersandneedleshavebeendeveloped considerably in the last fewdecades. In thefieldoftissueengineeringandregenerativemedicine,thereisaneedforadvancement over the conventional scaffolds and pre-formedhydrogels. In this scenario, injectable hydrogels have gainedwiderappreciationamongtheresearches,as theycanbeusedin minimally invasive surgical procedures. Injectable gels withtheireaseofhandling,completefillingthedefectareaandgoodpermeabilityhaveemergedaspromisingbiomaterials(Figure 5).

Basingontheirorigin,twokindsofbiodegradablepolymersareused for preparation of injectable hydrogels: naturally derivedand synthetic polymers. Naturally derived polymers such aspolysaccharides(cellulose,chitosan,hyaluronicacid,chondroitinsulfate,dextransulfate,alginate,pectin,etc.),proteins(collagen,

gelatin, heparin, fibrin, etc.) and synthetic polymers likepolypeptides, polyesters, polyphosphazenes, etc. are widelyused[178,179].Incomparisonwithsyntheticpolymers,mostbutnot all naturally derivedpolymers are expected to havebetterinteractionwithcellsalongwithincreasedcellproliferationanddifferentiation [180]. On the other hand, synthetic polymerspossesstunablemechanicalpropertiesanddegradationprofile.

Mutually,exclusiveadvantagesofthesepolymershavemotivatedtheresearcherstoinvestigatecombinatorialsystemsofsyntheticand naturally derived polymers, thereby improvising theproperties of injectable hydrogels [181]. Injectable hydrogelsare prepared using various physical and chemical crosslinkingmethodsasshowninFigure 6[182].

Particlemigration,poorshapedefinitionand/orrapidresorptionlimit thesuccessofcurrenturethralbulkingagents.Theuseofinjectable agents to augment urethral tissue function is nowcommon practice because they facilitate minimally invasivedeliveryofthebulkingagentandprovidetheadvantageof lowcostandlowpatientmorbidity.Theprocedurecanbeperformedinanoutpatientsettingwiththepatientunderlocalanesthesia.Current therapies using cross-linked collagen, autologous fator carbon coated beads have provided acceptable cure rates[183]. However, complications or limitations of the treatmentsincludetheneedforrepeatinjections,allergicreactions,materialresorptionandparticlemigration.Severalnewlyappliedmaterials,including calcium hydroxyapatite and materials consisting ofnaturalorsyntheticpolymersappeartobeefficaciousascurrenttherapiesbutextensivelong-termdataarestillunavailable[184].

Recent clinical trials as well as previous animal studiesdemonstrated that transplantationof autologous chondrocytessuspendedinapolymericalginatehydrogelproducedencouragingresultsandsuggestedthatinjectablealginatehydrogelsmayhavepotential use as bulking agents [185]. Covalently cross-linkedalginate hydrogels provide a versatile systemwith potential asan injectable bulking agent. The shapememory characteristicsofthesematerialsfacilitatedtheminimallyinvasivedeliveryofabulkingconstructinapredefinedthree-dimensionalmacroporousform,whichminimizedmigrationorleakageofthematerialfromthepuncturesiteandensuredintegrationwithhosttissue.Thehydrogelsareeasytoprepareandcanbestoreddehydratedatroomtemperature,requirednospecializedinjectionequipmentandcanbedeliveredbyasingle injection.Suchhydrogelshavethepotentialtofulfillmanyrequirementsforanoptimalurethralorothersofttissuebulkingagent.

InjectablehyaluronicacidMRIprovidesthepossibilitytomonitorthehydrogelandshapeofthematerialscorrectlybyonlyH-MRI.Therearemanyverypromisingstudies involvinghydrogelsandimagingtechniques.Inoneofthem,byYangX.andcollaborators,an injectable hyaluronic acid hydrogel has beenmodifiedwithfluorine-19andsubsequentlytested.Theauthorsclaimitshighpotential in many clinical applications since, besides it beingbiocompatibleandeasilyinjectable,itcanmoreoverbetrackedwithminimalinvasivemanners[186].

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Applications in electronicsTheuseofhydrogelsasmatrixesinelectronicsisverypromisingconsideringthehightunabilityandprecisionofcapacitorswithhydrogeldielectrics.By changing thepolymerand the solutioncarrieditispossibletobuildhigh-performantcheapcapacitors.

These Hydrogel electrolytes can be composed by organicpolymers. According to Choudhury et al. [187], poly(ethyleneoxide),potassiumpoly(acrylate),poly(vinyl alcohol) andgelatinareamongthemostpromisingmaterialsforthepurpose.However,organic polymer seem to show lower properties compared totheonesof inorganichydrogels.Manyauthorsaffirmthemoreinterestingcharacteristicofsilicahydrogelcomparedtostandardpolymerhydrogels,nevertheless,opinionsarestilldividedonthesubject.

ConclusionsInthisreviewweprovideahistoricaloverviewofthedevelopmentsin hydrogel research from simplenetworks to smartmaterials.Hydrogelsarewidelypresentineverydayproductsthoughtheirpotentialhasnotbeenfullyexploredyet.Thesematerialsalreadyhaveawell-establishedroleincontactlenses,hygieneproductsandwounddressingmarketsbutcommercialhydrogelproductsin tissue engineering and drug delivery are still limited.Manyhydrogel-based drug delivery devices and scaffold shave beendesigned, studied and in some cases even patented, howevernotmanyhavereachedthemarket.Moreprogressisexpectedinthesetwoareas.Limitedcommercialproductswithhydrogelsindrugdeliveryandtissueengineeringarerelatedtosomeextenttotheirhighproductioncosts.

Figure 5 Advantagesofinjectablehydrogelsoverconventionalscaffolds[179].

Figure 6 Schematicshowingdifferenttechniquestopreparehydrogels.

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Therearenumerouspatentsonhydrogelsbutonlyafewreachedthe market, hydrogels are used for producing contact lenses,hygieneproductsandwounddressings.Othercommercialusesofhydrogelsareindrugdeliveryandtissueengineering.

More developments are expected in drug delivery and tissueengineering,highproductioncostsofhydrogelsarelimitingtheirfurthercommercialization.

Over the past decades, significant progress has beenmade inthe field of hydrogels as functional biomaterials. Biomedicalapplication of hydrogels was initially hindered by the toxicityof crosslinking agents and limitations of hydrogel formationunderphysiologicalconditions.Emergingknowledgeinpolymer

chemistry and increased understanding of biological processesresultedinthedesignofversatilematerialsandminimallyinvasivetherapies.Hydrogelmatrices comprise awide rangeof naturalandsyntheticpolymersheldtogetherbyavarietyofphysicalorchemicalcrosslinks.Withtheircapacitytoembedpharmaceuticalagents in theirhydrophilic crosslinkednetwork,hydrogels formpromising materials for controlled drug release and tissue engineering.Despitealltheirbeneficialproperties,therearestillseveralchallengestoovercomeforclinicaltranslation.

AcknowledgmentsTheauthorsgratefullyacknowledgeMITACSprogramofQuébecandSarkoHoldingsforfinancialsupportofthiswork.

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