Injectable alginate hydrogels for cell delivery in tissue engineering

ReviewInjectable alginate hydrogels for cell delivery in tissue engineeringqSílvia J.Bidarra a ,⇑,Cristina C.Barrias a ,Pedro L.Granja a ,b ,caINEB –Instituto de Engenharia Biomédica,Universidade do Porto,Rua do Campo Alegre,823,4150-180Porto,PortugalbFEUP –Faculdade de Engenharia da Universidade do Porto,Departamento de Engenharia Metalúrgica e de Materiais,Rua Dr.Roberto Frias,s/n,4200-465Porto,Portugal cICBAS –Instituto de Ciências Biomédicas Abel Salazar,Universidade do Porto,Rua de Jorge Viterbo Ferreira,228,4050-313Porto,Portugala r t i c l e i n f o Article history:Available online 12December 2013Keywords:Tissue engineering Regeneration Cell delivery Injectable Alginatea b s t r a c tAlginate hydrogels are extremely versatile and adaptable biomaterials,with great potential for use in bio-medical applications.Their extracellular matrix-like features have been key factors for their choice as vehicles for cell delivery strategies aimed at tissue regeneration.A variety of strategies to decorate them with biofunctional moieties and to modulate their biophysical properties have been developed recently,which further allow their tailoring to the desired application.Additionally,their potential use as inject-able materials offers several advantages over preformed scaffold-based approaches,namely:easy incor-poration of therapeutic agents,such as cells,under mild conditions;minimally invasive local delivery;and high contourability,which is essential for filling in irregular defects.Alginate hydrogels have already been explored as cell delivery systems to enhance regeneration in different tissues and organs.Here,the in vitro and in vivo potential of injectable alginate hydrogels to deliver cells in a targeted fashion is reviewed.In each example,the selected crosslinking approach,the cell type,the target tissue and the main findings of the study are highlighted.Ó2013Acta Materialia Inc.Published by Elsevier Ltd.All rights reserved.1.Injectable cell delivery systemsIn many clinical scenarios,where normal physiological condi-tions or homeostasis are compromised,there is a need for tissue transplantation or implantation.The ideal paradigm in tissue engi-neering consists in introducing cells or tissues grafts,native to the injured area,to foster the regenerative process.In this context,cell-based therapeutic approaches can thus be considered a vital tool in regenerative medicine strategies.They rely on the successful deliv-ery of living cells to the target location,where they can produce a desired therapeutic effect by paracrine delivery of biomolecules (growth factors,cytokines,hormones,etc.)or replace lost cells with donor cells that can integrate and regenerate the damaged tissues [1,2].Cells used for cellular therapies can be previously manipulated to produce a missing substance,such as a specific protein that is absent in a metabolic disease [3].To ensure that an adequate number of cells reach the target tis-sue,cell-based approaches have usually been based on the delivery of high-density single-cell suspensions to the site of injury through injection.However,such direct cell injection often has a poor out-come due to large and rapid loss of cell viability (thus requiringhigh cell densities,which makes it technically complex and also extremely expensive),reduced engraftment of delivered cells and limited control over cell fate,in terms of both site-specificity (with cells eventually migrating and affecting other sites)and cell differ-entiation [4–6].Therefore,more effective cell transplantation methods,capable of sustaining the survival of implanted cells while maintaining their function and enhancing their incorpora-tion into the host,are mandatory.One strategy to achieve this goal relies on the delivery of transplanted cells via a temporary support made of biocompatible materials that can be further biochemically and physically modified to improve cell delivery [7,8].This strategy will provide cell protection,prolonged retention at the injury site and a more physiological three-dimensional (3-D)environment.Moreover,many studies have pointed to the importance of strate-gies that promote cell–cell and cell–matrix interactions,which im-pact considerably on cell morphology,viability and function [5,9].For instance,for anchorage-dependent cells,these interactions de-fine cell shape and organization,which in turn will regulate cell behavior,namely survival,differentiation,proliferation and migra-tion [10].Hydrogels are candidates of choice to mediate cell deliv-ery and accommodate cells in a 3-D microenvironment due to their natural similarities to the extracellular matrix (ECM).One paradig-matic example is alginate,a tunable and versatile natural inject-able hydrogel with huge potential as an artificial 3-D cellular matrix,which has already been explored in a myriad of studies as an injectable cell delivery system for a broad variety of biomed-ical applications [11].1742-7061/$-see front matter Ó2013Acta Materialia Inc.Published by Elsevier Ltd.All rights reserved./10.1016/j.actbio.2013.12.006qPart of the Special Issue on Biological Materials,edited by Professors Thomas H.Barker and Sarah C.Heilshorn⇑Corresponding author.Tel.:+351226074900;fax:+351226094567.E-mail addresses:sbidarra@ineb.up.pt (S.J.Bidarra),ccbarrias@ineb.up.pt (C.C.Barrias),pgranja@ineb.up.pt (P.L.Granja).For clinical applications,cell delivery through injectable materi-als may be a desirable method,since these systems offer specific advantages over preformed scaffold-based approaches.On the one hand,they can be applied using minimally invasive tech-niques,improving patient compliance and comfort,and eventually leading to faster recovery and hence lower healthcare costs.On the other hand,they present several additional appealing features, namely:(i)easy incorporation of therapeutic agents,such as pro-teins or cells,and their subsequent localized delivery;(ii)simplic-ity of implantation by injection;(iii)high contourability,providing adaptablefilling of defects with irregular shapes and sizes;and(iv) site specificity as well as confined delivery[12–22].A key requisite of cell delivery vehicles is the maintenance of cell viability throughout the injection procedure.For instance,whenflowing through a syringe needle,cells can experience three types of mechanical forces that can lead to cell disruption:(i)a pressure drop across the cell;(ii)shearing forces due to linear shearflow; and(iii)stretching forces due to extensionalflow[23].Therefore, an injectable matrix is also required to exhibit adequate mechani-cal properties to protect injected cells and thereby ensure their survival[23,24].Injectable cell-based systems can be prepared with different configurations,depending on the type of application.Distinct and often conflicting terms exist to designate these systems.In the present review,we propose a classification(Fig.1)in which sys-using a hydrogel-based vehicle[5].Although alginate hydrogels have been employed in all of these types of systems,this review will mainly focus on applications belonging to category3,thus the term‘‘entrapment’’will be used throughout the text.The main purpose of this review is to demonstrate the alginate´s potential as an adequate3-D microenvironment for cell delivery,in which cells are kept in direct contact with this synthetic ECM and,after trans-plantation,might become incorporated in the host tissue and ac-tively participate in the regeneration process.Therefore, categories1,2and4above will be described no further in this re-view.In strategy1,cells are actually in a2-D rather than a true3-D environment;strategy2aims at isolating cells from the host envi-ronment that will most likely not be incorporated in the host;and in strategy4the3-D environment is a result of the natural cellular aggregation and is not necessarily provided by the matrix.The present review focuses mainly on strategies where cell-la-den alginate-based systems were designed to be delivered in a minimally invasive way and,after injection,allow the transplanted cells to be integrated in the host’s damaged tissue and actively par-ticipate in the regeneration process.2.Hydrogels in tissue engineeringHydrogels are3-D hydrophilic,cross-linked polymeric net-immobilization strategies for injectable cell-based therapies.Several strategies and carrier materials can be used for cell immobilization.These caninjectable cell-based therapies.Here,the different approaches were divided into four major categories:(1)surface immobilization;(2)microencapsulation;and(4)multicellular aggregation.The bottom images correspond to injectable alginate-based systems incorporating cells.(A)Bone marrowdays on the surface of calcium titanium phosphate microspheres,under standard osteoinductive conditions.Cells were stained with Alexa-fluorcounterstained with propidium iodide(DNA).(B)Human mesenchymal stem cells(hMSCs)entrapped and cultured for21days inside2wt.%in basal conditions.(C)hMSCs entrapped and cultured for8days inside2wt.%PVGLIG/RGD–alginate hydrogels under basal conditions.Cells were488-phalloidin(F-actin)and counterstained with DAPI(DNA).(D)Mouse mammary epithelial cell line(EpH-4)within2wt.%RGD–alginate afterfor E-cadherin(red)and counterstained with DAPI(DNA).Adapted from Refs.[25,26].S.J.Bidarra et al./Acta Biomaterialia10(2014)1646–16621647compounds[14,24,34–40].Furthermore,many hydrogels can be formed under mild conditions,creating the adequate conditions for cytocompatible cell entrapment.In situ forming hydrogels present the added benefit of injecta-bility,and have been widely studied as cell carriers for in vivo tis-sue engineering[41].As previously pointed out,injectable hydrogels offer several advantages for cell entrapment and subse-quent delivery,for which purpose they should ideally combine a number of requisites,such as:(i)be in a sufficientlyfluid state dur-ing administration,like a low-viscosity solution,or undergo shear thinning before administration;(ii)gelation should start or be completed shortly after injection;(iii)be biocompatible and biode-gradable,and their products bioresorbable;and(iv)fulfill specific requirements according to the application envisaged(e.g.cell-adhesive capability for entrapment of anchorage-dependent cells).Injectable hydrogels can be obtained from either natural or syn-thetic polymers[42,43].Naturally derived polymers are frequently selected since these hydrogels either include components of the extracellular matrix(e.g.collagen,fribronectin,fribrinogen)or present a chemical structure similar to natural glycosaminoglycans (e.g.alginate,hyaluronic acid,chitosan),offering an intrinsic advantage over synthetic hydrogels[33].Since they are derived from natural sources,many of them contain cellular binding do-mains,thus allowing cell adhesion and/or present soluble signaling factors to become intrinsically bioactive and capable of regulate cellular behavior.However,they may exhibit batch-to-batch vari-ations and,in some cases,it is difficult to modulate their usually poor mechanical properties[33,44].Additionally,natural hydro-gels may be inherently immunogenic or give rise to some immuno-genicity due to the presence of contaminates,like proteins and endotoxins.In contrast with natural polymers,synthetic polymers allow better control over and reproducibility of their mechanical properties and chemistry[45].On the other hand,they may pres-ent a low degradation rate in physiological conditions and,in some cases,their preparation involves the use of toxic chemicals.Among these,polyethylene glycol(PEG)-based polymers are widely used as injectable hydrogels for cell delivery,and considerable efforts have been made in order to improve their features,including incor-poration of cell adhesion ligands or biodegradable units suscepti-ble to cell proteolytic activity,making them viable alternatives [38–40].3.Alginate hydrogelsAlginates are natural anionic biopolymers typically extracted from brown seaweeds.They are unbranched polysaccharides con-sisting of1,4-linked b-D mannuronic acid(M)and a-L-guluronic acid(G)units,which are covalently linked together in different number and sequence distributions along the polymer chain, depending on the alginate source[46].The functional properties of alginate depend on its monomer composition(M/G)ratio and sequence[47].For example,MG blocks(MGMGMGM)form the mostflexible chains and G blocks(GGGGGGG)form stiff chains. Moreover,alginate can be prepared with a wide range of molecular weights(typically101–103kDa)[24,47].Alginates are already used in several clinical applications,e.g. for treatment of heartburn and acid reflux(GavisconÒ,BisodolÒ, Asilone™),as wound dressing materials(AlgicellÒ,AlgiSite M™, ComfeelÒPlus,KaltostatÒ,SorbsanÒand Tegagen™)and as an appetite suppressant for long-term weight loss[48–50].Alginate is further being currently assessed in several clinical trials,namely as an agent for weight control,in the treatment of type I diabetes and as temporary acellular scaffolds to attenuate adverse cardiac remodeling[51].Additionally,tissue engineering alginate products are commercially available for3-D cell culture,such as AlgiMatrix from Invitrogen and NOVATACH peptide-coupled alginates from FMC Biopolymers.Alginate is considered to be non-immunogenic and has shown great potential as a cell delivery vehicle[46,50,52–54].In Fig.2 several examples of alginate as a cell delivery system are depicted. It is possible to visualize that alginate matrices allow the outward migration of different cell types,in this case human umbilical vein endothelial cells(HUVECs)(Fig.2A and B)and human mesenchy-mal stem cells(hMSCs)(Fig.2C and D),in different scenarios in vitro(Fig.2A),ex vivo(Fig.2B)and in vivo(Fig.2C and D).Cell migration from the matrix is a vital process when a cell delivery strategy is envisaged.As previously pointed out,an adequate vehicle must provide cell protection during injection.Alginate has been shown to have a shielding effect on cells during ejection from a syringe needle. Aguado et al.[23]tested1wt.%alginate with three different molec-ular weights on four different cell types(HUVECs,rat MSCs,human adipose stem cells and mouse neural progenitor cells),and have demonstrated the protective effect of alginate hydrogels with opti-mized mechanical properties(G0$30Pa).The authors showed that HUVECs in phosphate-buffered saline(PBS)or in non-crosslinked alginate have a significantly lower cell viability compared to in crosslinked alginates.Moreover,for all the studied cells,there was a significantly higher viability in alginate with G0$30Pa than in PBS or in alginate with a higher modulus.In an earlier study by Kong and colleagues[24],the authors showed that it is possible to maintain the alginate’s gel-forming ability by adjusting its molec-ular weight,while decreasing its viscosity and thus better preserv-ing cell viability(cell viability$80%within2and3.5wt.%alginate hydrogels).To obtain different molecular weights,the alginate was irradiated,resulting in solutions with different viscosities and deg-radation rates[47].Alginate presents a number of benefits for general use in bio-medical applications,coupled with a set of unique advantages for cell encapsulation and entrapment.Alginate allows the formation of hydrogels(with water)in situ,and the gelling process can be carried out using non-toxic solvents and under physiological con-ditions(namely in terms of pH and temperature),which provide easy cell encapsulation and entrapment.These hydrogels possess a soft nature,making them physically similar to most native tis-sues.Additionally,alginate mechanical properties can be tuned in order to encompass a range of stiffnesses that cover a variety of tissues.For example,the compression modulus of alginate hydrogels can range from1to1000kPa and the shear modulus from0.02to40kPa[55].Alginate mechanical properties can be controlled by changing different parameters,such as the polymer source,molecular weight,concentration and chemical modifica-tions,and/or the type and density of the crosslinking[46].Because they are transparent,alginate hydrogels allow the routine analysis of entrapped cells using standard microscopical techniques and they further enable easy cell recovery without cell damage. Regarding batch-to-batch variation and immunogenic response, both can be avoided through the use of highly purified and well-characterized alginates(concerning the molecular weight and the G/C ratio)that are currently commercially available.For instance, highly purified or ultrapure alginate contains low levels of residual endotoxin,lower than100EU gÀ1,and has been shown not to in-duce any severe foreign body reaction when implanted into ani-mals[53].As major drawbacks for these applications,alginate is slowly biodegradable and is not cell-interactive.However,these can be easily overcome by a number of chemical and biochemical modifications[14,15,35,56].Although alginate chains cannot be cleaved by mammalian en-zymes,ionically crosslinked alginate hydrogels disintegrate pro-gressively in vivo due to the exchange between monovalent cations,such as Na+,present in the surrounding milieu and the1648S.J.Bidarra et al./Acta Biomaterialia10(2014)1646–1662divalent cations that crosslink the hydrogel.Additionally,alginate can be modified to become degradable in physiological conditions by partial oxidation of alginate chains using sodium periodate [56].The periodate oxidation cleaves the carbon–carbon bond of the cis -diol group in the uronate residue and alters the chain conforma-tion to an upon-chain adduct,which behaves similar to an acetal group susceptible to hydrolysis.The degradation rate can be fur-ther regulated by adjusting the molecular weight distribution of oxidized alginates without varying the number of oxidized uronic acids,chain inflexibility and gel-forming ability [35].The molecu-lar weight can be modified by c -irradiation,which breaks the algi-nate chain [47].The oxidized binary hydrogels improved the formation of bone tissue compared to non-modified alginate,since a faster degradation occurs that facilitates the formation of new bone tissues [35].Additionally,alginates are chemically versatile,allowing the easy incorporation of biochemical cues to engineer specific cell responses.For example,as cells have no specific recep-tors for binding to alginate,several methods to promote cell attachment to alginate matrices have been developed.These in-clude the coupling of ECM proteins such as laminin,collagen and fibronectin [57–59].However,since the coupling of an entire pro-tein is difficult to control,can lead to non-specific interactions,may elicit an immune response and proteins are subject to proteolytic degradation [60],the decoration of biomaterials with cell recogni-tion motifs,such as small immobilized peptides,has become popular.The arginine–glycine–aspartic acid (RGD)sequence was one of the first peptides to be used to promote cell adhesion on a biomaterial and is still one of the most widely employed.This tri-peptide motif corresponds to the minimal essential cell adhesion peptide sequence identified in many ECM proteins,such as fibro-nectin,collagen,laminin,osteopontin and vitronectin,which are associated with integrins in cell surface membranes [60].To promote cell adhesion,alginate can be functionalized with this peptide using the aqueous carbodiimide chemistry,as demon-strated by Rowley et al.[34].In this case,RGD peptides are linked via a stable covalent amide bond and this reaction occurs between the carboxyl group of the alginate and the N-terminus of the peptide.Several other combined modifications have been investigated to improve alginate properties.For instance,the same chemical ap-proach was used to further tailor alginate hydrogels to a more sophisticated ECM-like 3-D cellular microenvironment.Alginate was grafted not only with RGD but also with a protease-labile crosslinking peptide (proline–valine–glycine–leucine–isoleucine–glycine,PVGLIG)that is cleavable by metalloproteinases (MMPs)produced by cells [14,15].The resultant MMP-sensitive hydrogels can be partially remodeled by cell-driven proteolytic mechanisms,leading to increased cellular evasion/invasion,and are particularly appealing vehicles for cell-delivery strategies.In another example,alginate was chemically functionalized with cell signaling moieties such as galactose to improve hepatocyte cell recognition [61,62].Since hepatocytes have a specific receptor that recognizes this li-gand,cells entrapped within alginate bearing galactose residues present higher functionality and survival rate.Also,to mimic the rheological behavior of the nucleus pulposus in the intervertebral disk,Leone et al.[63]have covalently crosslinked alginate by amide bond formation,after activation of the carboxylic groups and their conversion into amide moieties.They were able to attain a rheological behavior very similar to the one of the human nucleus pulposus and,when chondrocytes were added,they were able to proliferate and produce ECM proteins.According to the aim of the strategy,several other alginate modifications have been proposed to improve its behavior.Several alginate derivatives,their properties and possible applications are described in detail in a recent review by Pawar and Edgar [64].One of the greatest potentials of alginate is its mild and diverse gel-forming capacity.As described below,alginate hydrogels can be prepared by various crosslinking methods,which give rise to different delivery strategies with equally differentoutcomes.from alginate hydrogels.In vitro:HUVECs migrating out from an alginate–RGD hydrogel disk and (A)adhering tubular-like structures that sprout into Matrigel.In vivo:an hMSC-laden alginate–RGD–PVGLIG hydrogel after immunodeficiency (SCID)mice,showing (C)a paraffin section stained with safranin/light green (alginate stains in orange transplanted hMSC (arrows)can be observed within the disk,and (D)a paraffin section immunolabelled with an inside the hydrogel (dashed arrow)and another one at the hydrogel periphery (solid arrow),in close (⁄alginate;orange line –alginate matrix boundary).3.1.Formation of alginate hydrogelsAlginate hydrogels can be formed using chemical or physical crosslinking strategies,ionic methods being the most widely used. An overview of some of the crosslinking strategies that have been reported is presented in the following sections,and in Fig.3some of these strategies are represented schematically.3.1.1.Ionic crosslinkingThe most common method to achieve alginate gelation and crosslinking is through the exchange of sodium ions from guluron-ic acid units with divalent cations such as calcium(Ca2+),strontium (Sr2+)and barium(Ba2+)[67](Fig.3A).As alginates present differ-ent affinities towards the different divalent ions,they give rise to gels with different stability,permeability and strength,depending on the cation used[68].The stability of these hydrogels depends on the exchange between monovalent cations from the surrounding environment and the divalent cations from the hydrogel,resulting in diminished mechanical properties,which can limit their use for cell studies over long periods of time[48].This process depends on several alginate features,such as concentration,source,degree and type of crosslinking,and molecular weight[69].Moreover,the type of physiological conditions(in vitro or in vivo)will have an effect on hydrogel stability.Alginate stability is also variable in vitro. For instance,calcium-crosslinked alginate hydrogels rapidly lose stability in0.9wt.%sodium chloride,due to the exchange of cal-cium by non-gelling monovalent sodium ions[69],as well as in solutions containing chelators such as citrate or phosphate[70], which act as de-crosslinking agents by removing calcium ions.Cal-cium alginate hydrogels can nevertheless remain stable for weeks in cell culture medium,which generally contains sufficiently high calcium concentrations to counteract such effects[71].Ionic cross-linking of alginate can be further obtained by external or internal gelation.3.1.1.1.External gelation.External gelation of alginate hydrogels using soluble salts of divalent cations,such as calcium chloride (CaCl2),as ionic crosslinker agents is one of the most frequently used procedures,as it is a very simple process that provides imme-[17,19,20,73–95].These microbeads are generally produced by coaxial airflow,which controls the size of the droplets by blowing them from a needle tip into a CaCl2bath before they fall due to gravity.For a given alginate solution(concentration,type and vis-cosity),the size of the beads depends on the airflow,the needle diameter and solutionflow rate[48].To attain smaller beads ($150l m),other techniques must be used,such as electrostatic bead generation.In this case,a voltage is applied between the nee-dle and the electroconductive solution underneath[96].Therefore, it is possible to change the droplet size by adjusting the voltage. Nowadays,different types of microbead generators are commer-cially available.In a recent work,a novel methodology involving the use of superhydrophobic substrates was reported to efficiently produce spherical hydrogels entrapping rat MSCs isolated from bone mar-row andfibronectin without the need of a precipitation bath [97].Alginate beads were formed by applying microdrops of low-viscosity alginate onto superhydrophobic surfaces,then adding drops of CaCl2on top of each of them to crosslink the alginate.This methodology has a number of advantages over the conventional techniques,such as reduced mechanical forces and particle aggre-gation,which are coupled with decreased cell loss.Overall,alginate beads for tissue engineering should allow good permeability for nutrients and oxygen while providing optimal cell survival conditions for several days.Moreover,these beads allow thefixation of cells in the damaged tissue site,thus preventing cell removal via the bloodstream[97].Several examples in which alginate beads were used for tissue regeneration are discussed below.Although clearly demonstrating the enormous potential of alginate microbeads for cell entrapment, these works report the use of different conditions.Thus,the differ-ent alginate concentrations,molecular weights and even cell con-centrations used are highlighted.3.1.1.2.Internal gelation.Internal gelation strategies are being widely investigated with a view to promoting in situ hydrogel for-mation.This way,a polymeric solution combined with cells can be injected in a liquid state and will then form a hydrogel at the site of interest.Although these strategies have not been as extensively ex-alginate hydrogel formation.(A)Ionic crosslinking with calcium-induced chain–chain association of(egg-box model)[65].(B)Covalent crosslinking with PEG-diamines by carbodiimide chemistry.The hydrogelproperties:PEG chains provide some elasticity,while the alginate chains provide mechanical strength 1650S.J.Bidarra et al./Acta Biomaterialia10(2014)1646–1662ate(CaCO3)and calcium sulfate(CaSO4)have been widely used for this purpose.They have low solubility in pure water at neutral pH, but are soluble under acidic conditions,allowing its uniform distri-bution in the alginate solution before gelation occurs[72,100].Free calcium ions can later be released from these salts by slightly decreasing the pH with glucone-d-lactone(GDL),thereby allowing gradual gelation.In the case of CaCO3,the CaCO3/GDL molar ratio can be set at0.5to yield a neutral pH[14,72].It is notable that, although there is a slight decrease in pH,the cell viability is not af-fected[14,18].As an alternative to GDL,a photoacid generator(PAG)has been proposed that dissociates under UV light,releasing H+ions,which will react with CaCO3to create Ca2+[101].Despite the great poten-tial of these light-derived hydrogels as cell delivery systems for several biomedical applications,their cytocompatibility needs to be investigated further.Another way to promote photoactivated internal gelation consists in the use of water-soluble Ca2+chelators (‘‘cages’’),which can be mixed with alginate solutions and,upon light exposure,will undergo an irreversible molecular change that decreases their affinity to Ca2+.This will result in the release of Ca2+,which will subsequently trigger crosslinking[102].In con-trast to the use of insoluble calcium salts,this methodology allows homogeneous alginate hydrogels to be obtained even in concen-trated alginate solutions(e.g.10wt.%).Using this method, improvements in mechanical properties and homogeneity have been observed over comparable alginate concentrations.3.1.2.Covalent crosslinkingCovalent crosslinking of alginate hydrogels can be achieved by a variety of different methods,and usually provides more stable and mechanically stronger gels than ionic crosslinking.It is beyond the scope of the present article to review them all;instead,we focus on the strategies used for cell entrapment and delivery.Generally, once a material is covalently crosslinked,it no longer meets the injectability criteria.There are,however,a few exceptions,such as photocrosslinking alginates[103]and shape-memory alginate scaffolds[104],which will be explained later on.The major disad-vantages of these methods are their increased complexity and the eventual toxicity of the reagents used.Covalently crosslinked alginate hydrogels can be synthesized with a wide range of mechanical properties using,for example, PEG-diamine molecules with different molecular weights as cross-linkers[66](Fig.3B).The elastic modulus of the crosslinked algi-nate changes according to the molecular weight of the PEG molecules.The hydrogel properties can be further regulated by multifunctional crosslinking molecules,which provide a wider range and tighter control over degradation rates and mechanical stiffness,as demonstrated by Lee et al.[105].In their work,PAG hydrogels were formed with either poly(acrylamide-co-hydrazide) as a multifunctional crosslinking molecule or adipic acid dihydra-zide(AAD)as a bifunctional crosslinking molecule.This multi-crosslinking strategy led to the formation of stronger hydrogels.3.1.2.1.Photocrosslinking.Injectable photocrosslinkable alginates have been proposed for tissue engineering applications and have been designed to allow better control over mechanical properties, swelling ratios and degradation rates than ionically crosslinked alginates[50,103,106,107].Their cell-signaling ability can also be tailored by incorporating biochemical signals such as growth fac-tors and cell adhesive peptides[108,109].Photocrosslinkable alginate can be delivered in a minimally invasive way and then rapidly crosslinked in physiological condi-tions in situ following a brief exposure to ultraviolet(UV)light [110].This process occurs under mild conditions and therefore can be performed in direct contact with cells.For that purpose, alginate was modified with2-aminoethyl methacrylate using carbodiimide chemistry and these methacrylated alginates were subsequently photocrosslinked using UV light with a photoiniti-ator[103].Photocrosslinkable alginate with controlled degradation and cell adhesive properties have attracted great interest with re-gard ton tissue engineering applications[103,109,111,112], although they are still relatively new and seem to require further studies to assess their effectiveness.3.1.2.2.Shape-memory alginate scaffolds.A novel type of covalently crosslinked alginate,capable of retaining its shape and forming a macroporous structure,has been developed.The concept involves a hydrogel that allows control over its size and shape.Basically,a hydrogel is formed into the desired shape,then is collapsed in vitro for storage and handling purposes and,finally,is reformed in its original shape in vivo by rehydration[6,104].It is noteworthy that,although these covalently cross-linked alginate hydrogels present great potential as injectable bulking agents,they exhibit less contourability than other alginate hydrogels.On the other hand,they display greater shape definition,with a wider range of physical and mechanical properties.To obtain these shape-memory scaffolds,alginate was cova-lently crosslinked with AAD and the hydrogels were formed by standard carbodiimide chemistry using1-ethyl-(dimethyl amino-propyl)carbodiimide,1-hydroxybenzotriazole and AAD [104,113].Thornton et al.[104]were able to produce macroporous alginate hydrogel scaffolds with a predefined geometry,which were then dehydrated and compressed into small,temporary forms.The compressed scaffolds were then minimally invasively delivered to the dorsal subcutaneous space of a mouse through a catheter.Next,they were successfully rehydrated in situ by the injection of PBS.Overall,this work demonstrated the potential of these covalently crosslinked hydrogels as an injectable delivery system.More recently,Wang and colleagues[6]showed that these shape-memory scaffolds have the potential to serve as a synthetic matrix for skeletal muscle cell survival,proliferation and migra-tion.These results confirm the potential of these shape-memory alginate scaffolds as cell delivery systems for tissue regeneration, although this strategy needs further exploration.4.Alginate-based injectable cell delivery systems4.1.Bone regenerationIn thefields of orthopedics and oral and maxillofacial surgery, bone regeneration remains a clinical challenge,despite the intro-duction of various bone augmentation techniques and bone graft materials.The current standard treatment is based on the use of bone graft materials that are divided into two major groups:natu-ral and synthetic bone grafts[114].Natural bone grafts include auto-,allo-(human donors)and xenografts(other species).Autol-ogous bone graft is considered the‘‘gold standard’’for bone repair and regeneration by many surgeons,mainly due to lack of immu-nogenic reaction and optimal biological performance in terms of osteogenicity,osteoinductivity and osteoconductivity[115].How-ever,such bone grafts present several limitations,namely limited availability,the risks associated with harvesting,the additional surgical procedure required,donor site morbidity,post-operative pain and infection.On the other hand,allografts and xenografts are widely available and there is no need for additional surgery. However,with both types of graft there is the risk of immunoreac-tion and,since they undergo several processing techniques,their osteoinductive and osteoconductive potentials are reduced[115–117].To overcome the above-mentioned limitations of natural grafting,a large number of synthetic grafts have been designedS.J.Bidarra et al./Acta Biomaterialia10(2014)1646–16621651。