Review Hydrogels Based on Schiff Base Linkages for Biomedical Applications
1, 1, 1,2, Junpeng Xu †, Yi Liu † and Shan-hui Hsu * 1 Institute of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4 Roosevelt Road, Taipei 10617, Taiwan 2 Institute of Cellular and System Medicine, National Health Research Institutes, No. 35 Keyan Road, Miaoli 35053, Taiwan * Correspondence: [email protected]; Tel.: +886-2-3366-5313; Fax: +886-2-3366-5237 These authors contributed equally to this paper. † Received: 19 July 2019; Accepted: 13 August 2019; Published: 19 August 2019
Abstract: Schiff base, an important family of reaction in click chemistry, has received significant attention in the formation of self-healing hydrogels in recent years. Schiff base reversibly reacts even in mild conditions, which allows hydrogels with self-healing ability to recover their structures and functions after damages. Moreover, pH-sensitivity of the Schiff base offers the hydrogels response to biologically relevant stimuli. Different types of Schiff base can provide the hydrogels with tunable mechanical properties and chemical stabilities. In this review, we summarized the design and preparation of hydrogels based on various types of Schiff base linkages, as well as the biomedical applications of hydrogels in drug delivery, tissue regeneration, wound healing, tissue adhesives, bioprinting, and biosensors.
Keywords: Schiff base; hydrogel; click chemistry; self-healing; dynamic covalent bond; tissue engineering
1. Introduction Hydrogels with a three-dimensional structure and high water content are an important category of soft materials [1,2]. Hydrogels provide a physiologically similar environment for cell growth and they are often used to mimic the extracellular matrix (ECM) [3]. Cells interact and remodel with the ECM or ECM-like structures to achieve various cell behaviors, such as proliferation [4], migration [5], and differentiation [6]. Therefore, hydrogels have been developed for several biomedical applications [7], including tissue engineering [8], wound dressing [9], and drug delivery [10]. Hydrogels are generally formed by connecting hydrophilic polymer chains with various kinds of linkages to form networks [7]. The crosslinking network is divided into two types, i.e., the covalent network and non-covalent network, based on the nature of the linkages [11]. Click chemistry is one of the most common reactions used to obtain a covalent hydrogel network [12]. Click chemistry, initially proposed by Sharpless et al. in 2001, is a strategy combining various units to create highly selective products with a reliable high yield and effective atom utilization [13]. Click chemistry can yield smart materials that respond to external stimuli, and are often used as a key tool to prepare covalently crosslinked hydrogels for biomedical applications [14]. Typical examples of click chemistry are the Huisgen cycloaddition reaction [15], thiol-ene reaction [16], Diels–Alder reaction [17], Michael addition reaction [18], and the Schiff reaction [19]. These click reactions have been widely used in materials science [20–22], life sciences [23], and polymer chemistry [24,25]. Efficient click reactions in polymer chemistry have led to the development of new synthetic strategies. Click reactions can effectively create condensed polymers, as well as modify polymer chains and sophisticated architectures, including smart hydrogel networks or scaffolds. The Schiff reaction is a
Molecules 2019, 24, 3005; doi:10.3390/molecules24163005 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 22 Molecules 2019, 24, 3005 2 of 21 sophisticated architectures, including smart hydrogel networks or scaffolds. The Schiff reaction is a family of reactions used to generate dynamic Schiff base linkages, and it is currently one of the best methodsfamily of to reactions prepare usedsmart to biocompatible generate dynamic hydrogels. Schiff base linkages, and it is currently one of the best methodsThe toSchiff prepare reaction smart is biocompatiblea chemical reaction hydrogels. involving a dynamic covalent imine bond formation via theThe crosslinking Schiff reaction of amine is a chemical groups reactionand aldehyde involving groups a dynamic [26]. Schiff covalent base linkages imine bond can formationbe generated via inthe situ crosslinking with cells, of tissues, amine groupsand bioactive and aldehyde molecules groups under [26]. physiological Schiff base linkages conditions can beto generatedcreate hydrogel in situ withnetworks. cells, tissues,The dynamic and bioactive crosslinking molecules networks under can physiological also give hydrogels conditions their to create self-healing hydrogel properties networks. The[27].dynamic Moreover, crosslinking the Schiff networksbase is pH-responsive can also give hydrogels according their to its self-healing chemical structure properties [28]. [27]. Schiff Moreover, base linkages,the Schiff baseincluding is pH-responsive imines, hydrazones, according toand its oximes chemical, are structure the products [28]. Schi offf basecondensation linkages, includingreactions betweenimines, hydrazones, aldehyde groups and oximes, and various are the nucleophilic products of condensationamine groups. reactions Compared between to imines, aldehyde oximes groups and hydrazonesand various have nucleophilic better chemical amine groups. stability Compared with pH tovalue imines, changes oximes because and hydrazones the mesomeric have bettereffect reduceschemical the stability electrophilicity with pH of value the original changes carbon–nitrogen because the mesomeric double bond effect [28]. reduces Hydrogels the electrophilicity with various Schiffof the base original linkages carbon–nitrogen have unique double properties bond to [28 m].eet Hydrogels the requirements with various of different Schiff base tissues. linkages The Schiff have reactionunique propertieshas excellent to meet prospects the requirements in the biomedical of different field tissues.due to Theits simplicity, Schiff reaction reversibility, has excellent pH- prospectssensitivity, in and the biocompatibility. biomedical field due to its simplicity, reversibility, pH-sensitivity, and biocompatibility. Hydrogels with Schiff Schiff base linkages are currently used in several biomedical fields fields (Figure (Figure 11),), including drugdrug deliverydelivery [ 29[29,30],,30], tissue tissue regeneration regeneration [31 [31,32],,32], wound wound healing healing [33 ,[33,34],34], tissue tissue adhesives adhesives [35], [35],bioprinting bioprinting [36], and[36], biosensors and biosensors [37]. This [37]. review This review highlighted highlighted the use the of diusefferent of different Schiff base Schiff linkages base linkagesas crosslinking as crosslinking strategies strategies to prepare to biologically prepare biol relevantogically hydrogels relevant that hydrogels can be usedthat incan physiological be used in physiologicalenvironments. environments. After a brief classification After a brief of theclassification Schiff base, weof the summarized Schiff base, the chemicalwe summarized mechanisms the chemicalof different mechanisms linkages. The of preparationdifferent linkages. and biomedical The preparation applications and of hydrogelsbiomedical based applications on the Schi offf hydrogelsreaction were based further on the epitomized. Schiff reaction were further epitomized.
Figure 1. SummarySummary of of biomedical biomedical applications applications for for hydr hydrogelsogels based on various Schiff Schiff base linkages.
2. Design Design and and Preparation Preparation The Schiff Schiff base formation is obtained via the nucleophilicnucleophilic attack of amines on the electrophilic carbon atoms of aldehydes or keto ketones.nes. Hydrogels Hydrogels for biomedical applications have been extensively developed based on iminesimines andand theirtheir derivatives,derivatives, includingincluding hydrazoneshydrazones andand oximes.oximes. Imines, hydrazones, and oximes are formed by reaction between aldehydes/ketones aldehydes/ketones and primary amines, amines, hydrazides, and aminooxy, respectively. Hydrazones and and oximes exhibit better intrinsic stability compared to imines. Furthermore, Furthermore, acylhydrazones acylhydrazones allow allow higher higher hydrolytic hydrolytic stability compared to hydrazones and oximes [38]. [38]. In several recent st studies,udies, hydrazones and acylhydrazones with proper
Molecules 2019,, 24,, 3005x FOR PEER REVIEW 33 of of 2122 stability were employed to form crosslinks in hydrogel networks. Moreover, benzoic Schiff base stabilitylinkages werehave been employed widely to studied, form crosslinks where the in stab hydrogelility of networks. carbon–nitrogen Moreover, double benzoic bonds Schi hasff beenbase linkagesimproved have through been aromatic widely studied, substitution where with the stabilitybenzene ofrings carbon–nitrogen connected to doublethe carbon bonds or hasnitrogen been improvedatoms [39]. through Commonly, aromatic benzoic substitution Schiff base with linkages, benzene including rings connectedbenzoic imines, to the benzoic carbon hydrazides, or nitrogen atomsand benzoic [39]. Commonly, oximes, are benzoic formed Schi viaff linkagesbase linkages, of benzoic including aldehydes benzoic with imines, amines, benzoic hydrazides, hydrazides, and andaminooxy’s, benzoic respectively. oximes, are formed via linkages of benzoic aldehydes with amines, hydrazides, and aminooxy’s,Usually, respectively. chemical modifications are necessary to introduce aldehyde groups or nucleophilic groupsUsually, to the chemicalpolymers modifications for hydrogel arepreparations necessary tobased introduce on imine, aldehyde hydrazone, groups or or oxime nucleophilic linkages. groups The topolymers the polymers are functionalized for hydrogel preparationswith aldehyde based groups on imine, through hydrazone, two main or mechanisms: oxime linkages. (1) The The oxidative polymers arecleavage functionalized of vicinal with diols aldehyde with sodium groups periodate, through two and main (2) the mechanisms: conjugation (1) of The aldehyde-containing oxidative cleavage ofmolecules vicinal diolsusing with carbodiimide sodium periodate, chemistry. and Periodate-mediated (2) the conjugation oxidation of aldehyde-containing induces glycol cleavage molecules to usingdecompose carbodiimide into the aldehyde chemistry. products Periodate-mediated shown in Figure oxidation 2. Periodate-mediated induces glycol cleavage oxidation to decomposeis generally intoused theto aldehydecleave the products vicinal diols shown of invarious Figure polysa2. Periodate-mediatedccharides, such as oxidation alginate is[40–43], generally chondroitin used to cleavesulfate the[44], vicinal dextran diols [45–48], of various and hyaluronic polysaccharides, acid [49–51]. such as Carbodiimide alginate [40– chemistry43], chondroitin refers to sulfate amide [44 or], dextranester bond [45 formation–48], and hyaluronicof carboxylic acid acids [49 –and51]. amines Carbodiimide or hydroxyls chemistry with refersthe catalysis to amide of orcarbodiimide ester bond formationcompounds, of carboxylicsuch as water-soluble acids and amines 1-ethyl-3-(3-dimethylaminopropyl) or hydroxyls with the catalysis of carbodiimide compounds,(EDC) and suchwater-insoluble as water-soluble N,N’-dicyclohexylcarbodiimide 1-ethyl-3-(3-dimethylaminopropyl) (DCC). Aldehyde-containing carbodiimide (EDC) molecules, and water-insoluble such as 4- Nformylbenzoic,N0-dicyclohexylcarbodiimide acid, can be conjugated (DCC). Aldehyde-containing with the polymers molecules,through carbodiimide such as 4-formylbenzoic chemistry. acid,The canprimary be conjugated amine groups, with the as polymerstypical nucleophiles, through carbodiimide exist in several chemistry. natural The primaryand synthetic amine groups,polymers. as typicalHydrazide nucleophiles, groups are exist conjugated in several naturalwith polymers and synthetic through polymers. carbodiimide Hydrazide chemistry groups for are conjugatedhydrazone withformation, polymers whilst through aminooxy carbodiimide groups are chemistry conjugated for with hydrazone polymers formation, through whilst the Mitsunobu aminooxy reaction groups arefor conjugatedoxime formation. with polymers The Mitsunobu through reaction the Mitsunobu involves reaction the dehydrative for oxime coupling formation. of Thean alcohol Mitsunobu to a reactionpronucleophile, involves theincluding dehydrative carboxylic coupling acids, of an alcoholphenols, to aimides, pronucleophile, and sulfonamides, including carboxylic using acids,triphenylphosphine phenols, imides, and and an sulfonamides,azodicarboxylate. using triphenylphosphine and an azodicarboxylate.
Figure 2. Periodate-mediated oxidation inducing the cleavage of vicinal diols.
2.1. Imine-Based Hydrogels As shownshown in in Figure Figure3A, 3A, imines imines refer refer to compounds to compounds with carbon–nitrogenwith carbon–nitrogen double double bonds. Inbonds. several In studiesseveral studies regarding regarding imine-based imine-based hydrogels, hydrogels, polysaccharides polysaccharides (e.g., hyaluronic (e.g., hyaluronic acid, chondroitin acid, chondroitin sulfate, alginate,sulfate, alginate, and dextran) and dextran) are modified are modified with aldehyde with groupsaldehyde through groups periodate-mediated through periodate-mediated oxidation, andoxidation, then the and oxidized then the polysaccharides oxidized polysaccharides are used to form are imine-basedused to form hydrogels imine-based with varioushydrogels chitosan with derivativesvarious chitosan [41,42 derivatives,44,45,51,52 [41,42,44,45,51,52].]. Liu et al. reported Liu et the al. preparationreported the ofpreparation hydrogels of with hydrogels tough with and self-healingtough and self-healing properties properties through conjugation through conjugatio of oxidizedn of oxidized alginate alginate and acrylamide and acrylamide (AM) via (AM) imine via formationimine formation and subsequent and subsequent radical radical polymerization polymerization between between double bondsdouble on bonds conjugated on conjugated AM moieties AM andmoieties free AMand monomersfree AM monomers [43]. The hydrogels[43]. The hydrogels showed better showed self-healing better self-healing and mechanical and mechanical properties comparedproperties tocompared hydrogels to hydrogels prepared using prepared a mixture using ofa mixture oxidized of alginate oxidized and algi polyacrylamide.nate and polyacrylamide. Recently, benzoicRecently, imines benzoic have imines attracted have attentionattracted inattention biomaterials in biomaterials citing their citing self-healing their self-healing ability. We ability. introduce We theseintroduce researches these researches in the following in the sections.following sections.
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FigureFigure 3. FormationFormation of of imine, imine, hydrazone, hydrazone, acylhydrazone, acylhydrazo orne, oxime or oxime through through reactions reactions between between primary primaryamine, hydrazide, amine, hydrazide, acylhydrazide, acylhydr orazide, aminooxy or aminooxy and aldehyde. and aldehyde. 2.2. Hydrazone-Based Hydrogels 2.2. Hydrazone-Based Hydrogels As shown in Figure3B, hydrazones are formed by aldehydes /ketones and hydrazides. As shown in Figure 3B, hydrazones are formed by aldehydes/ketones and hydrazides. Figure Figure4A shows that hydrazide-functionalized polymers can be synthesized through conjugation of 4A shows that hydrazide-functionalized polymers can be synthesized through conjugation of tri-Boc- tri-Boc-hydrazinoacetic acid with amine groups on polymer chains and subsequent trifluoroacetic hydrazinoacetic acid with amine groups on polymer chains and subsequent trifluoroacetic acid acid deprotection [49,53,54]. Wang et al. prepared an injectable hydrogel based on dynamic covalent deprotection [49,53,54]. Wang et al. prepared an injectable hydrogel based on dynamic covalent hydrazone bonds between hydrazide-functionalized elastin-like protein and aldehyde-functionalized hydrazone bonds between hydrazide-functionalized elastin-like protein and aldehyde- hyaluronic acid (HA) [53]. Tri-Boc-hydrazinoacetic acid was conjugated with elastin-like protein functionalized hyaluronic acid (HA) [53]. Tri-Boc-hydrazinoacetic acid was conjugated with elastin- using a guanidinium (i.e., HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium like protein using a guanidinium (i.e., HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3- 3-oxid hexafluorophosphate) coupling reagent. Injectability to the hydrogel was provided by dynamic triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) coupling reagent. Injectability to the hydrazone crosslinking. Meanwhile, elastin-like protein thermal aggregates reinforced the hydrogel hydrogel was provided by dynamic hydrazone crosslinking. Meanwhile, elastin-like protein thermal with long-term degradation. McKinnon et al. reported an adaptable hydrogel that could respond to aggregates reinforced the hydrogel with long-term degradation. McKinnon et al. reported an cell-induced stress by rapidly breaking and reforming reversible hydrazone bonds [55,56]. Hydrogels adaptable hydrogel that could respond to cell-induced stress by rapidly breaking and reforming with a wide range of modulus and stress relaxation characteristics mimic the biophysics of native reversible hydrazone bonds [55,56]. Hydrogels with a wide range of modulus and stress relaxation tissue and allow the functions of natural cells. Hydrazone-based hydrogels with dynamic tunability characteristics mimic the biophysics of native tissue and allow the functions of natural cells. allow the development of cell morphologies, whereas non-adaptable hydrogels prevent cytoskeletal Hydrazone-based hydrogels with dynamic tunability allow the development of cell morphologies, rearrangement and extension. whereas non-adaptable hydrogels prevent cytoskeletal rearrangement and extension. Acylhydrazones, analogous to hydrazones, are formed by aldehydes/ketones and Acylhydrazones, analogous to hydrazones, are formed by aldehydes/ketones and acylhydrazides (Figure3C). Acylhydrazones are widely applied in the preparation of hydrogels acylhydrazides (Figure 3C). Acylhydrazones are widely applied in the preparation of hydrogels for for biomedical applications via the reaction between aldehyde-containing polymers and biomedical applications via the reaction between aldehyde-containing polymers and dihydrazide- dihydrazide-containing compounds or acylhydrazide-functionalized polymers [8,50,57–59]. Normally, containing compounds or acylhydrazide-functionalized polymers [8,50,57–59]. Normally, acylhydrazide-functionalized polymers are synthesized using carbodiimide chemistry between acylhydrazide-functionalized polymers are synthesized using carbodiimide chemistry between carboxyl-containing polymers and dihydrazide-containing compounds, such as carbodihydrazide carboxyl-containing polymers and dihydrazide-containing compounds, such as carbodihydrazide (CDH) and adipoyldihydrazide (ADH), similar to Figure4B. Wang et al. presented a shear-thinning (CDH) and adipoyldihydrazide (ADH), similar to Figure 4B. Wang et al. presented a shear-thinning and self-healing hydrogel based on dynamic acylhydrazone bonds for three-dimensional (3D) and self-healing hydrogel based on dynamic acylhydrazone bonds for three-dimensional (3D) printing [50]. In their study, acylhydrazine-functionalized HA was prepared via carbodiimide printing [50]. In their study, acylhydrazine-functionalized HA was prepared via carbodiimide chemistry between carboxyl groups on HA and amine groups on ADH. The hydrogels, formed by chemistry between carboxyl groups on HA and amine groups on ADH. The hydrogels, formed by mixing acylhydrazine-functionalized HA and aldehyde-functionalized HA, could be extruded and mixing acylhydrazine-functionalized HA and aldehyde-functionalized HA, could be extruded and printed as 3D constructs with high fidelity and stability. printed as 3D constructs with high fidelity and stability.
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Figure 4. (A) Synthesis of hydrazide-functionalized polymer using carbodiimide chemistry coupling
Figurewith tri-Boc-hydrazinoacetic 4. (A) Synthesis of hydrazide-functionalized acid for amide formation, polymer and using subsequent carbodiimide trifluoroacetic chemistry acid coupling (TFA) Figure 4. (A) Synthesis of hydrazide-functionalized polymer using carbodiimide chemistry coupling withdeprotection tri-Boc-hydrazinoacetic of tert-butoxycarbonyl acid for (Boc) amide groups. formation, (B) Synthesis and subsequent of acylhy trifluoroaceticdrazide-functionalized acid (TFA) with tri-Boc-hydrazinoacetic acid for amide formation, and subsequent trifluoroacetic acid (TFA) deprotectionpolymer using of tertcarbodiimide-butoxycarbonyl chemistry (Boc) coupling groups. with (B) Synthesis dihydrazide-containing of acylhydrazide-functionalized compounds, for deprotection of tert-butoxycarbonyl (Boc) groups. (B) Synthesis of acylhydrazide-functionalized polymerexample, using adipoyldihydrazide carbodiimide chemistry coupling coupling with a carboxyl with dihydrazide-containing group by amide formation. compounds, for example, polymer using carbodiimide chemistry coupling with dihydrazide-containing compounds, for adipoyldihydrazide coupling with a carboxyl group by amide formation. 2.3. Oxime-Basedexample, adipoyldihydrazide Hydrogels coupling with a carboxyl group by amide formation. 2.3. Oxime-Based Hydrogels 2.3. Oxime-BasedOxime is obtained Hydrogels through a reaction between aldehydes/ketones and hydroxylamine, as shown in FigureOxime 3D. is obtainedOxime-based through hydrogels a reaction can betweenbe formed aldehydes using aminooxy-functionalized/ketones and hydroxylamine, polymers as shown and Oxime is obtained through a reaction between aldehydes/ketones and hydroxylamine, as shown inaldehyde-functionalized Figure3D. Oxime-based polymers. hydrogels Aminooxy-functionalized can be formed using aminooxy-functionalized polymers can be synthesized polymers in andtwo in Figure 3D. Oxime-based hydrogels can be formed using aminooxy-functionalized polymers and aldehyde-functionalizedsteps using the Mitsunobu polymers. reaction, coupling Aminooxy-functionalized N-hydroxyphthalimide polymers with canhydroxyl be synthesized on the polymers, in two aldehyde-functionalized polymers. Aminooxy-functionalized polymers can be synthesized in two stepsfollowed using by the deprotection Mitsunobu with reaction, hydrazine coupling (FigureN-hydroxyphthalimide 5) [8,47,60–64]. Grover with hydroxylet al. reported on the on polymers, oxime- steps using the Mitsunobu reaction, coupling N-hydroxyphthalimide with hydroxyl on the polymers, followedbased injectable by deprotection hydrogels with using hydrazine aminooxy-functionalized (Figure5)[ 8,47,60– 64four-arm]. Grover poly(ethylene et al. reported oxide) on oxime-based (AO-PEO) followed by deprotection with hydrazine (Figure 5) [8,47,60–64]. Grover et al. reported on oxime- injectableand ketone-functionalized hydrogels using aminooxy-functionalizedfour-arm poly(ethylene oxide) four-arm (Ket-PEO) poly(ethylene [60]. For oxide) the synthesis (AO-PEO) of AO- and based injectable hydrogels using aminooxy-functionalized four-arm poly(ethylene oxide) (AO-PEO) ketone-functionalizedPEO, triphenylphosphine four-arm and poly(ethylenediisopropyl azodicarboxylate, oxide) (Ket-PEO) respectively, [60]. For the synthesiswere added of AO-PEO,into the and ketone-functionalized four-arm poly(ethylene oxide) (Ket-PEO) [60]. For the synthesis of AO- triphenylphosphinemixture of alcohol-terminated and diisopropyl four-arm azodicarboxylate, PEO and N-hydroxyphthalimide respectively, were in added the cold into condition. the mixture After of PEO, triphenylphosphine and diisopropyl azodicarboxylate, respectively, were added into the alcohol-terminateda one day reaction, four-armthe product PEO was and mixedN-hydroxyphthalimide with hydrazine monohydrate. in the cold condition. The hydrogels After awith one pH- day mixture of alcohol-terminated four-arm PEO and N-hydroxyphthalimide in the cold condition. After reaction,tunable gelation the product could was be mixed formed with hydrazineby AO-PEO monohydrate. and Ket-PEO, The as hydrogels well as withAO-PEO pH-tunable and oxidized gelation a one day reaction, the product was mixed with hydrazine monohydrate. The hydrogels with pH- couldhyaluronic be formed acid byor AO-PEOoxidized andalginate. Ket-PEO, Recently, as well photomediated as AO-PEO and oxime oxidized linkages hyaluronic were acid developed or oxidized for tunable gelation could be formed by AO-PEO and Ket-PEO, as well as AO-PEO and oxidized alginate.spatiotemporally Recently, controlled photomediated hydrogel oxime formation linkages were[65,66]. developed In these for researches, spatiotemporally 2-(2-nitrophenyl) controlled hyaluronic acid or oxidized alginate. Recently, photomediated oxime linkages were developed for hydrogelpropyloxycarbonyl formation (NPPOC) [65,66]. In was these utilized researches, as a direct 2-(2-nitrophenyl) photocage for propyloxycarbonyl aminooxys. Upon UV (NPPOC) irradiation, was spatiotemporally controlled hydrogel formation [65,66]. In these researches, 2-(2-nitrophenyl) utilizedthe aminooxys as a direct were photocage freed from for the aminooxys. NPPOC photocag Upon UVes irradiation, and they reacted the aminooxys with aldehydes were freed to fromform propyloxycarbonyl (NPPOC) was utilized as a direct photocage for aminooxys. Upon UV irradiation, theoxime NPPOC linkages. photocages Photomediated and they oxime reacted linkages with aldehydes provided tocontrol form oximeover the linkages. location Photomediated and extent of the aminooxys were freed from the NPPOC photocages and they reacted with aldehydes to form oximecrosslinking linkages for provided the systematic control tuning over the of location mechanical and extentproperties. of crosslinking Moreover, for photomediated the systematic tuningoxime oxime linkages. Photomediated oxime linkages provided control over the location and extent of oflinkages mechanical could properties. be applied Moreover, to immobilize photomediated various biomolecules oxime linkages with could spatiotemporal be applied to control immobilize and crosslinking for the systematic tuning of mechanical properties. Moreover, photomediated oxime variousmicron-scale biomolecules resolution. with spatiotemporal control and micron-scale resolution. linkages could be applied to immobilize various biomolecules with spatiotemporal control and micron-scale resolution.
Figure 5. Synthesis of aminooxy-functionalized polymers using the Mitsunobu reaction coupling with Figure 5. Synthesis of aminooxy-functionalized polymers using the Mitsunobu reaction coupling with hydroxyphthalimide, followedfollowed byby deprotectiondeprotection withwith hydrazinehydrazine (N(N2H 4). Figure 5. Synthesis of aminooxy-functionalized polymers using the Mitsunobu2 4 reaction coupling with 2.4. Benzoichydroxyphthalimide, Schiff Base-Based followed Hydrogels by deprotection with hydrazine (N2H4).
2.4. Benzoic Schiff Base-Based Hydrogels
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2.4. Benzoic Schiff Base-Based Hydrogels The benzoic Schiff base has attracted sizable attention in the preparation of hydrogels for biomedical applications. In addition to higher stability, the benzoic Schiff base gives hydrogels enhanced self-healing properties compared to an aliphatic Schiff base. Benzaldehyde-containing small molecules, such as zinc phthalocyanine tetra-aldehyde (ZnPcTa) [67,68] and 3,5-diformyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3,5-diformyl-BODIPY) [69], have been developed as crosslinkers to generate benzoic Schiff base-based hydrogels. In most cases, benzaldehyde-functionalized polymers are synthesized to react with polymers containing amine groups. Benzaldehyde-terminated poly(ethylene oxide) (PEO) can be prepared through a nucleophilic substitution reaction followed by the synthesis of mesylate-terminated PEO, similar to Figure6A [10,70,71]. First, mesylate-terminated PEO is synthesized through the reaction of PEO, trimethylamine, and methanesulfonyl chloride in methylene chloride solution. Then, 4-hydroxybenzaldehyde and potassium carbonate are added to the solution of mesylate-terminated PEO in dimethylformamide to obtain benzaldehyde-terminated PEO. Deng et al. reported a dynamic hydrogel with an environmental adaptive self-healing property via acylhydrazone linkages between benzaldehyde-terminated three-arm PEO and dithiodipropionic acid dihydrazide [59]. The hydrogel could automatically repair damages under acidic conditions through acylhydrazone exchange, whilst the hydrogel was not self-healable at pH 7 due to kinetically locked bonds. However, addition of catalytic aniline to the hydrogel facilitated hydrogel self-healing at pH 7 by accelerating acylhydrazone exchange. Yu et al. designed an injectable self-healing hydrogel based on a chain extended Pluronic F127 (PEO90-PPO65-PEO90) multi-block copolymer [71]. Benzaldehyde-terminated Pluronic F127 was synthesized and then extended using adipic dihydrazide via the formation of acylhydrazone bonds. The hydrogel exhibited rapid sol-gel transition at body temperature based on the thermo-responsivity of Pluronic F127. Moreover, dynamic acylhydrazone bonds allowed the hydrogel to recover its mechanical properties and structure after repeated damage. Benzaldehyde-terminated PEO can also be facilely prepared in one step using carbodiimide condensation chemistry, similar to Figure6B[ 37,72–76]. The very first work on the synthesis of benzaldehyde-terminated Pluronic L64 using carbodiimide chemistry was reported in 2010 [75]. The injectable hydrogels based on benzaldehyde-terminated Pluronic L64 and glycol chitosan were prepared under dual pH- and temperature-responsiveness. The benzaldehyde-terminated PEO has been widely used to form injectable self-healing hydrogels for various biomaterial applications. Benzaldehyde-terminated telechelic PEO was synthesized via the conjugation of 4-formylbenzoic acid with PEO chain ends catalyzed by DCC and 4-(dimethylamino)pyridine (DMAP) [73]. Benzaldehyde-terminated PEO was used to form a self-healing hydrogel with chitosan through dynamic Schiff base linkages. Moreover, the hydrogels showed multi-responsive behavior to pH, amino acids, and vitamin B6 derivatives because of the dynamic equilibrium between the Schiff base linkage and the aldehyde and amine reactants. Khan et al. prepared a benzaldehyde-terminated four-arm PEO using four-arm PEO and 4-formylbenzoic acid via an EDA coupling reaction [74]. An injectable self-healing hydrogel was generated using benzaldehyde-terminated four-arm PEO and chitosan-g-l-glutamic acid. The hydrogels exhibited tunable mechanical properties by varying the benzaldehyde-terminated four-arm PEO concentration or the total solid content of the hydrogels. As shown in Figure6C, a novel photogenerated aldehyde group (i.e., 2-nitrobenzyl alcohol group) has been developed to functionalize the polymers using carbodiimide chemistry [54,77,78]. 2-nitrosobenzaldehyde moieties can be converted by 2-nitrobenzyl alcohol moieties exposed to a 365 nm light wavelength. After generation of the benzaldehyde upon UV radiation, the phototriggered imine-crosslinked hydrogels would rapidly form. Yang et al. prepared a phototriggered imine-crosslinked hydrogel using hyaluronic acid-nitrobenzyl alcohol (HA-NB) and hyaluronic acid-carbohydrazide (HA-CDH) [77]. In this study, a 2-nitrobenzyl alcohol-containing molecule (i.e., N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide) was synthesized to conjugate with HA for the preparation of photoreactive HA-NB. The hydrogel allowed excellent Molecules 2019, 24, 3005 7 of 21 integration with surrounding tissue after in situ phototriggered crosslinking, owing to the imine Moleculesformation 2019 between, 24, x FOR photogenerated PEER REVIEW aldehydes on HA-NB and amines on the tissue surface. 7 of 22
A
B
C
FigureFigure 6. 6. SynthesisSynthesis of of benzaldehyde-functi benzaldehyde-functionalizedonalized polymers polymers through through ( (AA)) a a nucleophilic nucleophilic substitution substitution reactionreaction after after the the conversion conversion of of an an alcohol alcohol to toa me a mesylate;sylate; or carbodiimide or carbodiimide chemistry chemistry coupling coupling with with the molecularthe molecular containing containing benzaldehyde benzaldehyde groups groups (B) (orB )2-nitrobenzyl or 2-nitrobenzyl alcohol alcohol groups groups (C). ( CPhotoreactive). Photoreactive 2- nitrobenzyl2-nitrobenzyl alcohol alcohol can can convert convert to to2-nitr 2-nitrosobenzaldehydeosobenzaldehyde upon upon UV UV irradiation. irradiation.
3.3. Biomedical Biomedical Applications Applications HydrogelsHydrogels used used in in the the biomedical field field have several requirements, such such as as formation under under physiologicalphysiological conditions, conditions, rapid rapid crosslinking, crosslinking, and and biocompatibility. biocompatibility. In In several several studies, studies, click click chemistry, chemistry, suchsuch as as the the Huisgen cycloaddition reaction, reaction, thiol- thiol-eneene reaction, reaction, Diels–Alder Diels–Alder reaction, reaction, and and the the Schiff Schiff reaction,reaction, has has been been developed developed to to form crosslinks in hydrogel networks. However, However, metal metal catalysts, catalysts, radicalradical initiators,initiators, andand poor poor reactivity reactivity tend tend to limit to limit the Huisgen the Huisgen cycloaddition cycloaddition reaction, reaction, thiol-ene thiol-ene reaction, reaction,and Diels–Alder and Diels–Alder reaction, respectivelyreaction, respectively [79]. Compared [79]. Compared to other click to reactions,other click the reactions, Schiff reaction the Schiff can reactionrapidly proceedcan rapidly under proceed mild conditions under mild without conditio metalns catalysts.without Moreover,metal catalysts. the Schi Moreover,ff reaction the is used Schiff in reactionbiomaterials is used due toin itsbiomaterials reversibility, due pH-responsiveness, to its reversibility, high pH-responsiveness, reactivity, and biocompatibility. high reactivity, A Schi andff biocompatibility.reaction with dynamic A Schiff equilibrium reaction with naturally dynamic exists equilibrium as one of thenaturally organisms exists self-healing as one of the mechanisms. organisms self-healingIn this section, mechanisms. we focused In on this the section, various we applications focused on of the hydrogels various withapplications Schiff base of hydrogels linkages inwith the Schiffbiomedical base linkages field. in the biomedical field.
3.1. Drug Delivery 3.1. Drug Delivery OneOne of of the the most most important important applications applications of of hydrogels hydrogels is is drug drug delivery. delivery. Chemotherapy Chemotherapy is is clinically clinically usedused against against tumors tumors and and cancer, cancer, although challenges remain, remain, such such as inaccurate drug drug delivery and and highhigh initial initial dosage. dosage. Hydrogels Hydrogels are are potential potential candidates candidates to to improve improve accuracy accuracy and and efficiency, efficiency, as as well asas reduce reduce the the adverse adverse effects effects of chemotherapy. As As drug carriers, hydrogels hydrogels should should have have appropriate appropriate mechanicalmechanical properties,properties, injectability,injectability, biodegradability, biodegradability, and and biocompatibility. biocompatibility. Particularly, Particularly, carriers carriers must mustrelease release the drug the drug in a temporospatiallyin a temporospatially appropriate appropriate manner. manner. Hydrogels Hydrogels with with Schi Schiffff base base linkages linkages are aresuitable suitable to be to designed be designed and developed and developed to satisfy to applicationsatisfy application on various on tissuesvarious citing tissues their citing possibility their possibilityto meet the to aforementioned meet the aforementioned requirements. requirements. GlycolGlycol chitosan-basedchitosan-based hydrogels hydrogels crosslinked crosslinked with difunctionalized with poly(difunctionalizedN-isopropylacrylamide)- poly(N- isopropylacrylamide)-co-poly(acrylicco-poly(acrylic acid) were prepared as acid) drug were delivery prepared vehicles asfor drug anticancer delivery chemotherapy vehicles for anticancer [80]. Such chemotherapyhydrogels were [80]. designed Such hydrogels to be injectable, were self-healable,designed to be pH-sensitive, injectable, self-healable, and temperature-sensitive. pH-sensitive, and The temperature-sensitive.release rate of cisplatin The from release such hydrogels rate of cisplati was promotedn from such in thehydrogels acidic condition was promoted when thein the structure acidic conditionof the hydrogels when the collapsed. structure The of controlledthe hydrogels drug collap releasesed. behavior The controlled of the hydrogels drug release in the behavior corresponding of the hydrogelspH environment in the suggestedcorresponding that thesepH environment hydrogels could suggested be used that in drugthese delivery.hydrogels could be used in drug delivery. A self-healing hydrogel based on glycol chitosan and difunctionalized polyethylene glycol (DF- PEG) was loaded onto Taxol for antitumor therapy (Figure 7A) [81]. Compared to Pluronic F127 hydrogels, the injectable hydrogel released the drug for a significantly longer period. Tumor
Molecules 2019, 24, x FOR PEER REVIEW 8 of 22 xenograft in nude mice also confirmed a better antitumor effect of the self-healing hydrogel compared to the drug only control group and the drug-loaded Pluronic F127 hydrogel (Figure 7B,C) (the animal experiments were performed in accordance with protocols approved by the ethics committee of Cancer Hospital, Chinese Academy of Medical Science). The results suggested that a self-healing hydrogel was good at the encapsulation and controlled release of drugs. Another advantage of hydrogels with Schiff base linkages is that different chemicals can be conveniently loaded, considering that in vivo chemotherapies often involve the efficient synergistic effect of several drugs. For example, a dual-drug-loaded magnetic hydrogel (DDMH) with thermosensitivity and self-healing properties was prepared using glycol chitosan and DF-PEG for tumor therapy [82]. Doxorubicin (DOX), iron oxide, and docetaxel (DTX) were carried in the DDMH. DDMH could exhibit the potential for stimuli-responsive drug release and magnetic hyperthermia. DDMH proved to have good biocompatibility and asynchronous controlled release properties in cell experiments. At the same time, DDMH showed excellent thermal induction performance under an alternative magnetic field (AMF) to control the surrounding temperature (Figure 7D). The results of in vitro and in vivo antitumor experiments showed that the synergistic antitumor effect of hydrogels loaded with two chemicals was significantly enhanced compared to DOX or DTX/PLGA nanoparticle-loaded hydrogels, respectively (Figure 7E,F1,F2). In addition, the asynchronous controlled release of DOX and DTX and the controlled release by AMF-trigger in synchronous drug deliveryMolecules systems2019, 24, 3005 were more effective in anticancer chemotherapy. 8 of 21 Embolization agents are essential for the treatment of various unresectable malignant tumors. A three-component dynamic self-healing hydrogel based on glycol-chitosan, carbazochrome, and DF- PEG wasA self-healing recently prepared hydrogel to basedovercome on glycolthe limit chitosanation of and low difunctionalized therapeutic efficiency polyethylene [30]. The glycol controlled(DF-PEG) release was loaded of carbazochrome onto Taxol for and antitumor the tunabl therapye decomposition (Figure7A) of [ 81hydrogels]. Compared were tofound Pluronic to be F127 achievedhydrogels, using the DF-PEG injectable with hydrogel various released concentrations. the drug The for a three-component significantly longer hydrogels period. could Tumor change xenograft thein nudecrosslinking mice also density confirmed of the a betterinternal antitumor structure e ffandect ofcontrol the self-healing the release hydrogel rate of carbazochrome compared to the by drug increasingonly control the group concentration and the drug-loadedof DF-PEG. The Pluronic self-hea F127ling hydrogel hydrogel (Figure could7 effectivelyB,C) (the animal treat the experiments target embolization,were performed and init had accordance a low cost with and protocols risk for in approved vivo use. by Additionally, the ethics committee the size and of gelation Cancer Hospital,time ofChinese the hydrogel Academy could of Medicalbe precisely Science). controlled The results to potentiate suggested the thathydrogel a self-healing for targeting hydrogel embolization was good at therapy.the encapsulation and controlled release of drugs.
FigureFigure 7. 7. (A(A) Schematics) Schematics of ofthe the intratumor intratumor experi experimentment through through hydrogel hydrogel injection. injection. (B) Results (B) Results of the of the intratumorintratumor injection injection in innude nude mice mice divided divided into into four four groups groups according according to different to different injection injection treatments, treatments, including only injected saline (Blank), only injected Taxol solution (Tax H O), injected Pluronic F127 · 2 hydrogel with Taxol (Tax F127), and injected self-healing hydrogel based on glycol chitosan and DF-PEG · with Taxol (Tax CP) (the animal experiments were performed in accordance with protocols approved · by the ethics committee of Cancer Hospital, Chinese Academy of Medical Science). (C) The volume and appearance of tumors in mice in the four groups. Reprinted with permission, as in Reference [81]. © 2017, Royal Society of Chemistry (London, UK). (D) Infrared thermal images for tumor-bearing mice under exposure to an AMF at different post-injection treatments (saline and DDMH) (the animal experiments were performed in accordance with protocols approved by the Laboratory Animal Research Center, Tsinghua University). (E) Tumor growth curves with different treatments (only saline, only DOX solution, DTX solution with PLGA nanoparticles, DOX solution with PLGA nanoparticles, and DDMH solution with AMF). (F1,F2) The temperature of different tumor sites shown during the exposure. Reprinted with permission [82]. © 2015, American Chemical Society (Washington, DC, USA).
Another advantage of hydrogels with Schiff base linkages is that different chemicals can be conveniently loaded, considering that in vivo chemotherapies often involve the efficient synergistic effect of several drugs. For example, a dual-drug-loaded magnetic hydrogel (DDMH) with thermosensitivity and self-healing properties was prepared using glycol chitosan and DF-PEG for tumor therapy [82]. Doxorubicin (DOX), iron oxide, and docetaxel (DTX) were carried in the DDMH. DDMH could exhibit the potential for stimuli-responsive drug release and magnetic hyperthermia. DDMH proved to have good biocompatibility and asynchronous controlled release properties in cell experiments. At the same time, DDMH showed excellent thermal induction performance Molecules 2019, 24, 3005 9 of 21 under an alternative magnetic field (AMF) to control the surrounding temperature (Figure7D). The results of in vitro and in vivo antitumor experiments showed that the synergistic antitumor effect of hydrogels loaded with two chemicals was significantly enhanced compared to DOX or DTX/PLGA nanoparticle-loaded hydrogels, respectively (Figure7E,F1,F2). In addition, the asynchronous controlled release of DOX and DTX and the controlled release by AMF-trigger in synchronous drug delivery systems were more effective in anticancer chemotherapy. Embolization agents are essential for the treatment of various unresectable malignant tumors. A three-component dynamic self-healing hydrogel based on glycol-chitosan, carbazochrome, and DF-PEG was recently prepared to overcome the limitation of low therapeutic efficiency [30]. The controlled release of carbazochrome and the tunable decomposition of hydrogels were found to be achieved using DF-PEG with various concentrations. The three-component hydrogels could change the crosslinking density of the internal structure and control the release rate of carbazochrome by increasing the concentration of DF-PEG. The self-healing hydrogel could effectively treat the target embolization, and it had a low cost and risk for in vivo use. Additionally, the size and gelation time of the hydrogel could be precisely controlled to potentiate the hydrogel for targeting embolization therapy.
3.2. Wound Healing Over the last century, hydrogels have been the focus of wound healing materials. Hydrogels can absorb and retain wound exudates, and at the same time, promote fibroblast proliferation and wound epithelium formation [83]. The internal structure of the hydrogel is very compact (the hole being 100 nm in full swelling state), which can effectively prevent bacteria from entering the wound and keep the wound moist [84,85]. Hydrogels have unique and adjustable mechanical properties to fit wounds of different tissues [86]. Compared to conventional bandages or gauzes, the high water retaining capacity of hydrogels makes them particularly soothing on wounds, and the non-sticky nature of hydrogels causes less discomfort in patients and less pain during peeling [87]. As a coolant for local wounds, it can also relieve pain and restore the degree of damage. Recently, quaternized chitosan-g-polyaniline (QCSP) and benzaldehyde group functionalized poly(ethylene glycol)-co-poly(glycerol sebacate) (PEGS-FA) solution were mixed to prepare injectable self-healing hydrogel dressing [34]. Compared to traditional commercial dressings, the self-healing hydrogel dressing had better antibacterial activity, electric activity, and free radical scavenging ability (Figure8A,B). Besides, it was possible to adjust the gelation process to regulate the rate of gelation, pore size, electrical conductivity, and swelling ratio of the self-healing hydrogel. Simultaneously, hemostatic properties could be obtained by adjusting the ratios of different QCSP and PEGS-FA. The efficiency of such hydrogels in repairing full-thickness skin wounds has been confirmed in animal experiments (Figure8C). Self-adaptive self-healing hydrogels based on chitosan and DF-PEG have been successfully developed [72]. Given the dynamic Schiff bases, the hydrogels can change their shape and move freely. Human internal tissues have difficulty in responding to external stimuli, while traditional intelligent biomaterials hardly change their shape and location to adapt to the internal oral environment. Hydrogels show better therapeutic effects in wound healing in vivo experiments relative to other control treatments, supporting the self-adapting effect of the materials on wound healing. Hydrogels can also carry drugs and act in synergy with their properties on wound healing. A novel injectable hydrogel loaded with nano-curcumin based on N,O-carboxymethyl chitosan (CCS) and oxidized alginate (OA) was reported to accelerate skin wound repair [88]. Compared to commercial dressings and unloaded hydrogel controls, the nano-curcumin/CCS-OA hydrogel significantly decreased the wound area, and complete wound closure occurred after 14 days of treatment (Figure8D,E). In vivo and in vitro experiments also confirmed that nano-curcumin could be released from the proposed hydrogel to enhance epidermal re-epithelialization and collagen deposition in wound tissue. Molecules 2019, 24, 3005 10 of 21
Injectable hydrogels of chitosan and oxidized konjac glucomannan designed using the Schiff reaction mechanism were successfully produced [9]. These injectable hydrogels had self-healing ability Moleculesand biocompatibility, 2019, 24, x FOR PEER which REVIEW could benefit wound healing. The physical properties (such as porosity10 of 22 and swelling ratio) could be changed by adjusting the crosslinking density. Meanwhile, excellent externalinhibition bacterial rates of Staphylococcusinfections during aureus theand healingEscherichia process. coli Thecould injected prevent hydrogel wounds significantly from external shortened bacterial theinfections wound during healing the time healing using process. a full-thickness The injected skin hydrogel defect significantly model test. shortened Additionally, the wound histological healing examinationstime using a full-thickness showed that skinthe result defectant model hydrogel test. Additionally, significantly histologicalaccelerated examinationsthe re-epithelialization showed that of damagedthe resultant tissues. hydrogel significantly accelerated the re-epithelialization of damaged tissues.
Figure 8. ((AA)) Photographs Photographs of of wounds wounds at at the the 0th, 0th, 5th, 5th, 10t 10th,h, and and 15th 15th day, day, and and granulation granulation tissue tissue on the on 15ththe 15th day dayfor forcommercial commercial film film dressings dressings (Tegader (Tegadermm™),™ ),hydrogel hydrogel QCS/PE QCS/PEGS-FA,GS-FA, and and hydrogel QCSP/PEGS-FA.QCSP/PEGS-FA. (B)) WoundWound contractioncontraction forfor thethe threethree groups.groups. (C) Granulation tissue thickness for the three groups onon thethe 15th15th day.day. ReprintedReprinted withwith permissionpermission [[34]34].. ©© 2017, Elsevier Ltd.Ltd. (Amsterdam, TheNetherlands). Netherlands). (D) (PhotographsD) Photographs of wound of wound treated treated over over 14 14 days days with with saline saline solution, solution, blank blank CCS-OA CCS-OA hydrogel, unmodified unmodified curcumin loaded CCS-OA hydrogel, and nano-curcumin/CCS-OA nano-curcumin/CCS-OA hydrogel. hydrogel. (E) The average wound area on three rats in eacheach group (the animal experiments were performed in accordance withwith protocols protocols approved approved by theby Institutionalthe Institutional Animal Animal Care and Care Use and Committee Use Committee of Wenzhou of Wenzhoumedical college). medical Reprinted college). with Reprinted permission with [88 permission]. © 2012, Elsevier [88]. © Ltd. 2012, (Amsterdam, Elsevier Ltd. The Netherlands).(Amsterdam, Netherlands). 3.3. Tissue Regeneration 3.3. TissueHydrogels Regeneration that can be injected into the body to promote tissue repair have become a popular topic, and theyHydrogels have also that found can be several injected tissue into engineering the body to applications promote tissue in recent repair years have [ 89become]. Hydrogels a popular are topic,considered and they to be have biocompatible also found becauseseveral tissue of their engineering structural similaritiesapplications with in recent macromolecules years [89]. Hydrogelsin vivo [7]. areHydrogels considered can alsoto be mimic biocompatibl many propertiese because ofof the their extracellular structural simi matrixlarities in tissues, with ma suchcromolecules as regulating in vivocell functions [7]. Hydrogels and permitting can also mimic the diff manyusion properties of nutrients, of metabolites,the extracellular and growthmatrix factorsin tissues, [90 ].such Using as regulatinginjectable hydrogels cell functions allows and clinicians permitting to transplant the diffus correspondingion of nutrients, cells metabolites, in a minimally and invasive growth manner, factors [90].and toUsing a certain injectable extent, hydrogels promote tissueallows regeneration. clinicians to transplant corresponding cells in a minimally invasiveSelf-healing manner, hydrogelsand to a certain based extent, on glycol promote chitosan tissue and regeneration. DF-PEG have been employed as a means to µ improveSelf-healing neural tissue hydrogels regeneration based on [31 glycol]. Neural chitosan stem celland spheroids DF-PEG have with anbeen appropriate employed size as a ( Molecules 2019, 24, 3005 11 of 21 An injectable chitosan-fibrin (CF) hydrogel with an interpenetrating polymer network can be prepared by adding fibrin into a glycol chitosan-based hydrogel [32]. Vascular endothelial cells in CF Moleculeshydrogels 2019 may, 24, x form FOR capillary-likePEER REVIEW structures. The injection of CF hydrogel alone promoted angiogenesis11 of 22 in the perivitelline space of zebrafish (Figure9A). The CF hydrogel also proved to successfully repair successfullythe defects in repair ischemic the defects hindlimb in ischemic mice, as hindlimb well as rescuing mice, as blood well as circulation rescuing (Figureblood circulation9B,C) (the (Figure animal 9B,C)experiments (the animal were performedexperiments in accordancewere performed with protocols in accordance approved with by prot theocols University approved Animal by Carethe Universityand Use Committee). Animal Care and Use Committee). AddingAdding conductive conductive polymers polymers to to hydrogels hydrogels is is a a common common way way to to generate generate conductive conductive hydrogels, hydrogels, whichwhich can benefitbenefit electroactive electroactive cells cells and and tissues. tissues. The The combination combination of dextran-graft-aniline of dextran-graft-aniline tetramer-graft- tetramer- graft-4-formylbenzoic4-formylbenzoic acid (Dex-AT)acid (Dex-AT) and Nand-carboxyethyl N-carboxyethyl chitosan chitosan (CECS) (CECS) hydrogels hydrogels was was performed performed as a asconductive a conductive injectable injectable self-healing self-healing hydrogel hydrogel [91]. [91]. Given Given the dynamicthe dynamic action action of the of Schi theff Schiffbase, base, these theseconductive conductive hydrogels hydrogels had good had injectability good injectabil and degradability.ity and degradability. L929 fibroblasts L929 couldfibroblasts proliferate could in proliferatethe hydrogel. in the C2C12 hydrogel. myoblasts C2C12 and myoblasts human umbilical and human vein umbilical endothelial vein cells endothelial that were cells grown that inwere the grownhydrogel in couldthe hydrogel be released could from be thereleased hydrogel from matrix the hydrogel with a linear matrix profile, with anda linear the released profile, cellsand stillthe releasedshowed cells an ability still showed for continuous an ability proliferation. for continuous Compared proliferation. to the Compared PBS control to the group PBS and control Dex /groupCECS andhydrogel, Dex/CECS the regeneration hydrogel, the of regeneration skeletal muscle of skel usingetal a Dex-ATmuscle /usingCECS a hydrogel Dex-AT/CECS was confirmed hydrogel inwas an confirmedanimal model in an within animal four model weeks, within as shown four weeks, in Figure as shown9D,E. in Figure 9D,E. FigureFigure 9. 9. ((AA)) The The pattern pattern of of the the sub sub intestinal intestinal vessels vessels (S (SIV)IV) in in zebrafish zebrafish embryos embryos in in which which all all of of the the vascularvascular endothelial endothelial cells cells expressed expressed enhanced enhanced green green fluorescent fluorescent protein. Each Each embryo embryo was was injected injected withwith PBS, PBS, CS CS hydrogel hydrogel or or CF CF hy hydrogeldrogel without cells cells 24 24 h after fertilization, and and the the fluorescence fluorescence microscopicmicroscopic images images were were taken taken one one day day after after the the injection. injection. ( (BB)) Representative Representative images images of of the the ischemic ischemic hindhind limbs limbs of of mice mice injected injected with with PBS PBS or or CF CF hydrogel hydrogel (the (the animal experiments were were performed performed in in accordanceaccordance with with protocols protocols approved approved by by the the Univ Universityersity Animal Animal Care Care and Use Committee). The The PBS PBS groupgroup suffered suffered severe severe nail loss. No No intramedullary intramedullary nail nail fell fell off off inin the the CF CF hydrogel hydrogel group group and and limb limb salvagesalvage was was successful. successful. (C (C) )The The blood blood flow flow ratios ratios of of ischemic ischemic limbs limbs and and healthy healthy limbs limbs in in different different groupsgroups showed showed a a significant significant increa increasese in in blood blood flow flow after after surgery surgery in in the the hydrogel hydrogel injection injection group. group. ReprintedReprinted with permission [[32]32].. ©© 2017,2017, NatureNature PublishingPublishing Group Group (London, (London, UK). UK). The The regeneration regeneration of ofskeletal skeletal muscle muscle tissue tissue was was evaluated evaluated using using H&E H&E staining staining in in aa ratrat skeletalskeletal muscle volume loss model model afterafter volumetric musclemuscle loss loss injury injury and and treatment treatment for for 1 week 1 week (D) and(D) 4and weeks 4 weeks (E) between (E) between three groups, three groups,including including PBS control, PBS control, Dex/CECS Dex/CECS hydrogel hydrogel treatment, treatment, and Dex-AT and Dex-AT/C/CECS hydrogelECS hydrogel treatment treatment (black (blackarrows: arrows: centronucleated centronucleated myofibers; myofibers; yellow yellow arrows: arrows: newly newly formed formed blood blood vessels). vessels). Reprinted Reprinted with withpermission permission [91]. [91]© 2019,. © 2019, Elsevier Elsevier Ltd. Ltd. (Amsterdam, (Amsterdam, The Netherlands).Netherlands). Cell therapy is a potential strategy for tissue regeneration in heart disease. To effectively carry Cell therapy is a potential strategy for tissue regeneration in heart disease. To effectively carry cells, biocompatible hydrogels can be a proper candidate for cell therapy. A conductive hydrogel cells, biocompatible hydrogels can be a proper candidate for cell therapy. A conductive hydrogel based on chitosan-graft-aniline tetramer (CS-AT) and DF-PEG was prepared [92]. The conductivity based on chitosan-graft-aniline tetramer (CS-AT) and DF-PEG was prepared [92]. The conductivity of the prepared hydrogel was close to that of natural heart tissue, and it showed good tissue of the prepared hydrogel was close to that of natural heart tissue, and it showed good tissue adhesion adhesion and antimicrobial activity to the target tissue. C2C12 myoblasts and H9c2 cells encapsulated and antimicrobial activity to the target tissue. C2C12 myoblasts and H9c2 cells encapsulated in such in such hydrogels determined good viability and proliferation. Meanwhile, the injectability and hydrogels determined good viability and proliferation. Meanwhile, the injectability and biodegradability of hydrogels were proven through subcutaneous injection and in vivo degradation. biodegradability of hydrogels were proven through subcutaneous injection and in vivo degradation. Collectively, the experimental results showed that the hydrogel was an excellent cell carrier for cardiac tissue regeneration. N-succinyl-chitosan (S-CS) and aldehyde-hyaluronic acid (A-HA) based composite hydrogels were successfully prepared, and they showed good biocompatibility [93]. As the ratio of S-CS to A- HA increased, the compressive modulus of the composite hydrogel improved, and the in vitro degradation rate slightly increased. The S-CS/A-HA hydrogels may promote cell adhesion and Molecules 2019, 24, x FOR PEER REVIEW 12 of 22 proliferation, as well as maintain regular cell morphology of bovine articular chondrocytes, demonstrating potential applications in cartilage tissue engineering. Molecules 2019, 24, 3005 12 of 21 3.4. Bioprinting Collectively,Regarding the experimentalthe manufacturing results process showed thatfor tissue the hydrogel engineering was an applications, excellent cell combining carrier for cardiacthree- dimensionaltissue regeneration. (3D) bioprinting technology with bioink, a substance containing living cells and materialsN-succinyl-chitosan to support the adhesion, (S-CS) and proliferation, aldehyde-hyaluronic and differentiation acid (A-HA) of cells based can generate composite 3D hydrogels scaffolds withwere a successfully tissue-like structure. prepared, The and tissue-like they showed 3D goodscaffold biocompatibility can provide structural [93]. As the support ratio of for S-CS cells to at A-HA both aincreased, micro- and the macro-level compressive to modulus improve of the the characteri compositestics hydrogel of the growth improved, environment and the in vitro[94]. Meanwhile,degradation suchrate slightly3D scaffolds increased. can be Thedesigned S-CS/ A-HAusing computer hydrogels programming may promote and cell controlling adhesion andprinting proliferation, parameters as towell achieve as maintain the requirements regular cell morphology of high resolution, of bovine multi-length articular chondrocytes, scale, and demonstratingcustomization potential[95]. 3D bioprintingapplications has in cartilage been extensively tissue engineering. studied in the biomedical field citing in situ cell mixing, rapid manufacturing, and the potential for artificial vessels and organs. 3.4. BioprintingIn recent years, hyaluronic acid-based hydrogels have been successfully prepared to contain dynamicRegarding hydrazone the bonds manufacturing using modified process hyaluronic for tissue acid with engineering hydrazide applications, or aldehyde groups combining [50]. Thesethree-dimensional hydrogels can (3D) be used bioprinting to print technology 3D structures with with bioink, high ashape substance fidelity, containing relaxation living stability, cells and cellmaterials compatibility to support due the to adhesion,the shear thinning proliferation, and self-repairing and differentiation properties of cells of canhydrogels generate with 3D dynamic scaffolds bondswith a (Figure tissue-like 10A). structure. The extruded The tissue-like hydrogel 3D filaments scaffold printed can provide with structural cells in situ support have a for smooth cells atsurface, both a highmicro- quality and macro-level of reproduction, to improve and no the relaxation characteristics in the of printing the growth process environment (Figure 10B). [94]. Further Meanwhile, complex such structures3D scaffolds can can be besuccessfully designed usingprinted computer with high programming cell viability (Figure and controlling 10C). printing parameters to achieveAldehyde-containing the requirements of oxidized high resolution, alginate multi-length was recently scale, developed and customization as a base material [95]. 3D bioprintingto develop self-repairinghas been extensively and 3D studiedbioprintable in the hydrogels biomedical with field different citing kinds in situ of cell crosslinking mixing, rapid agents manufacturing, (Figure 10D) [36].and theDifferent potential imine for artificialdynamic vesselscovalent and bonds organs. (oxime, semicarbazide, and hydrazone) were used to controlIn recentthe hardness, years, hyaluronic viscoelasticity, acid-based self-healing hydrogels properties, have been 3D successfullyprintability, preparedand morphological to contain changesdynamic of hydrazone cells in bondsthe hydrogels. using modified Compared hyaluronic to oxime-bonded acid with hydrazide hydrogels, or aldehyde semicarbazide groups and [50]. hydrazone-bondedThese hydrogels can hydrogels be used to are print viscoelastic, 3D structures self with-healable, high shapeand 3D fidelity, bioprintable. relaxation Subsequently, stability, and humancell compatibility dermal fibroblasts due to the in shear printed thinning hydrogels and self-repairing kept growing properties over seven of hydrogels days. The with viscoelastic dynamic crosslinkingbonds (Figure (urea 10A). and The extrudedhydrazone) hydrogel was beneficial filaments printedto cell withextension cells inand situ growth, have a smooth whilst surface,elastic crosslinkinghigh quality of(oxime) reproduction, restricted and the no morphology relaxation inof thehuman printing dermal process fibroblasts (Figure to 10 remainB). Further in a complexcircular shape.structures can be successfully printed with high cell viability (Figure 10C). Figure 10. ((AA)) Schematics Schematics for for shear-thinning shear-thinning and and self-healing self-healing of of hydrogel hydrogelss during during printing, printing, where where the hydrogels were formed in syringes syringes,, and filaments filaments were stabilized by the self-healing properties of hydrazone bonds through shearing during the extrusion process. ( (BB)) Photos Photos of of 4-layer 4-layer lattices lattices in in air air and in PBS. ( C) Live/dead Live/dead staining of cells in lattice filamentsfilaments immediately after extrusion. Reprinted with permission [50] [50].. © 2018, Wiley-VCH (Weinheim, Germany). ( D) The schematic diagram for the chemical structure and advantages of the bioprintable hydrogels [36]. chemical structure and advantages of the bioprintable hydrogels [36]. Aldehyde-containing oxidized alginate was recently developed as a base material to develop self-repairing and 3D bioprintable hydrogels with different kinds of crosslinking agents (Figure 10D) [36]. Different imine dynamic covalent bonds (oxime, semicarbazide, and hydrazone) were used to control the hardness, viscoelasticity, self-healing properties, 3D printability, and morphological changes of Molecules 2019, 24, 3005 13 of 21 cells in the hydrogels. Compared to oxime-bonded hydrogels, semicarbazide and hydrazone-bonded hydrogels are viscoelastic, self-healable, and 3D bioprintable. Subsequently, human dermal fibroblasts in printed hydrogels kept growing over seven days. The viscoelastic crosslinking (urea and hydrazone) was beneficial to cell extension and growth, whilst elastic crosslinking (oxime) restricted the morphology of human dermal fibroblasts to remain in a circular shape. Water-soluble hydroxybutyl chitosan (HBC) and chondroitin oxysulfate (OCS) were used to prepare bioink based on the Schiff-base reaction to print different structures of hydrogels using different sacrificial molds with 3D bioprinting technology [96]. After the formula optimization, the injectable hydrogels with uniform internal holes (100 µm) were printed to form macroporous hydrogels. The biocompatible hydrogel constructs with various internal and external structures promoted the viability of cultured human adipose-derived mesenchymal stem cells. The bionic HBC/OCS hydrogels with controllable shape could be optimized and customized for specific cartilage engineering applications. Therefore, bioprintable HBC/OCS hydrogel has applications in tissue engineering. 3.5. Tissue Adhesives With the rapid growth of the wound care market, tissue adhesives have been well studied and developed. In surgery, tissue adhesives can be used to replace conventional surgical adhesives due to their sufficient tensile strength, short bonding time, simple usage, and no need for postoperative removal [97]. Furthermore, the use of tissue adhesives in organisms needs to consider tissue toxicity given direct contact with body fluids or blood. Meanwhile, adhesives need to show the corresponding physical and chemical properties for different regions and tissues, fast adherence to target sites, and rapid hemostasis [35,98]. Therefore, biomedical adhesives have more stringent conditions related to toxicity and harmfulness, as well as strict biocompatibility and biodegradability standards. Hydrogels, as materials with excellent potential for tissue engineering, can be designed to achieve the aforementioned conditions for biomedical tissue adhesives across various applications by employing different materials and formulations. Previously, a double crosslinked network (DN) hydrogel based on the combination of HA and furan derivatives using a Diels–Alder (DA) click reaction and acylhydrazone bond was successfully prepared [57]. Dynamic covalent acylhydrazone bonds can impart hydrogels with self-healing properties and control the crosslinking density of the network. Aldehyde groups in hydrogels based on the Schiff reaction can promote the adhesion of hydrogels to surrounding tissues. In contrast to non-sticky DA hydrogels, push-out tests were used to determine the bonding strength between DN hydrogels and cartilage. The results showed that the bond strength of the target hydrogel (10.3 0.7 kPa) was significantly higher than that of the DA hydrogel (1.2 0.5 kPa), which supported ± ± the tissue adhesive ability of the DN hydrogel. In recent years, injectable double crosslinked self-healing hydrogels based on dopamine-grafted oxidized sodium alginate (OSA-DA) and polyacrylamide (PAM) have been reported for wound healing (Figure 11A) [99]. Given the hydrogen bond and Schiff base bond, the OSA-DA-PAM self-healing hydrogel has stable mechanical properties, such as high tensile strength (0.109 MPa) and elongation (2550%). Additionally, a large number of catechol groups on the OSA-DA chain may endow the hydrogel with unique cell affinity and tissue adhesion. The above properties of OSA-DA-PAM hydrogels are demonstrated in Figure 11B,C using animal experiments with robust wound protection and the ability to promote tissue regeneration. A self-healing injectable micellar hydrogel can be successfully prepared (Figure 11D) under physiological conditions using quaternized chitosan (QCS) and benzaldehyde-terminated Pluronic F127 (PF) [70]. QCS/PF hydrogels exhibit appropriate modulus that is similar to human soft tissue, as well as stable rheological properties, excellent cell adhesion, and biocompatibility. Simultaneously, the resultant hydrogel also has pH response characteristics showing a high drug release rate in an acidic skin environment. The inherent antimicrobial property, free radical scavenging ability, and good coagulation ability of the hydrogel can effectively promote wound healing. Moreover, animal experiments validated Molecules 2019, 24, 3005 14 of 21 the faster healing ability of the QCS/PF hydrogel compared to commercial wound adhesive dressings (TegadermTM). Besides, QCS/PF hydrogel loaded with curcumin (Cur-QCS/PF hydrogel) significantly promotes wound healing by up-regulating the production of wound healing-related factors and down-regulatingMolecules 2019, 24, x FOR the PEER production REVIEW of pro-inflammatory factors. Cur-QCS/PF hydrogel can also14 of be22 applied to the human elbow citing its tissue adhesive ability Hydrogels based on aminated star-shaped polyethylenepolyethylene glycol and aldehyde-dextran polymers have been reported as tissuetissue adhesivesadhesives [[100].100]. HydrogelsHydrogels with various cohesiveness, adhesiveness, and other physical properties can be designed by controlling the aldehydealdehyde/amine/amine ratio, which can produce materials that are adhesive to specificspecific tissues.tissues. The adhesive hydrogel has also shown good biocompatibility and tissue stickiness during in vivo andand inin vitrovitro studies.studies. Two-component hydrogelshydrogels based based on aldehyde-dextranon aldehyde-dextran and dendritic and dendritic polyamidoamine polyamidoamine dendrimer weredendrimer successfully were preparedsuccessfully as tissueprepared adhesive as tissue materials adhesive [101]. materials The viscous [101] hydrogel. The providesviscous hydrogel viscosity viaprovides the crosslinking viscosity via between the crosslinking aldehyde between and amine aldehyde groups, and and amine it produces groups, an and adhesive it produces interface an inadhesive vivo by interface reacting within vivo histamine by reacting glucan with aldehyde. histamine During glucan material aldehyde. design, During the interface material morphology, design, the bondinginterface strength,morphology, and bonding mechanicalstrength, and properties bonding ofmechanical the hydrogel properties can be adjustedof the hydrogel by controlling can be theadjusted aldehyde by /aminecontrolling ratio tothe achieve aldehyde/amine the specific microenvironment ratio to achieve conditions.the specific Good microenvironment biocompatibility, tunability,conditions. andGood tissue biocompatibility, adhesion were supportedtunability, inand cell tissue experiments adhesion and were mouse supported duodenal models.in cell (Figureexperiments 11E). and mouse duodenal models. Figure 11. ((AA)) The The schematic schematic diagram diagram of of tissue tissue adhesive adhesive hydrogels. (B) Optical Optical images of wound sites were taken on days 0, 5, and 15 after surgery. (C)) RemainingRemaining woundwound ratiosratios forfor eacheach group.group. Reprinted with permission [[99]99].. ©© 2018, American American Chemical Soci Societyety (Washington, DC, USA). (D) The schematic illustration of the Cur-QCS/PF Cur-QCS/PF hydr hydrogelogel and the transmissi transmissionon electron microscope image of of PF (i.e., PF127-CHO) micelles. Scale Scale bar: bar: 200 200 nm. nm. ( (EE)) The The photograph photograph of of Cur-QC Cur-QCSS/PF/PF hydrogels that were applied on the human elbow. Scale bar: 5 cm. Reprinted with with permission permission [70] [70].. © 2018,2018, Elsevier Elsevier Ltd. Ltd. (Amsterdam, TheNetherlands). Netherlands). 3.6. Biosensors 3.6. Biosensors Hydrogels, as a self-adhesive and elastic soft material with great potential in biomedical Hydrogels, as a self-adhesive and elastic soft material with great potential in biomedical applications, can be used for wearable applications, including electronic skin and biosensors after being applications, can be used for wearable applications, including electronic skin and biosensors after endowed with electrical conductivity [102]. Conductive hydrogels are typically based on conductive being endowed with electrical conductivity [102]. Conductive hydrogels are typically based on polymers with intrinsic electron conductivity, as well as conductive nanomaterials, such as polypyrrole conductive polymers with intrinsic electron conductivity, as well as conductive nanomaterials, such (PPy), polythiophene (PT), polyaniline (PANI), carbon nanotubes, and metallic nanoparticles [103]. as polypyrrole (PPy), polythiophene (PT), polyaniline (PANI), carbon nanotubes, and metallic These different conductive hydrogels act on different fields depending on their strength, electrical nanoparticles [103]. These different conductive hydrogels act on different fields depending on their conductivity, and stimuli responsiveness. In recent years, biosensors have been vigorously developed. strength, electrical conductivity, and stimuli responsiveness. In recent years, biosensors have been A dynamic covalent hydrogel, based on an imine bond prepared using human neutrophil elastase vigorously developed. (HNE), Ala-Ala-Pro-Val-Ala-Ala-Lys, and aldehyde-dextran was successfully reported [104]. In this A dynamic covalent hydrogel, based on an imine bond prepared using human neutrophil study, hydrogel membranes were prepared by coating, which led to rapid selective degradation elastase (HNE), Ala-Ala-Pro-Val-Ala-Ala-Lys, and aldehyde-dextran was successfully reported [104]. when enzymes were added. In the presence of periodontal disease markers, the activity of HNE and In this study, hydrogel membranes were prepared by coating, which led to rapid selective degradation when enzymes were added. In the presence of periodontal disease markers, the activity of HNE and cathepsin could be directly measured by monitoring the degradation of the membranes. The low enzyme concentration could also be detected by increasing the crosslinking density of the peptide to increase the degradation rate (i.e., sensitivity). A conductive hydrogel based on graphene oxide, dopamine, and polyacrylamide was prepared using the Schiff reaction, as shown in Figure 12 [37]. The high stretchability, toughness, and self- Molecules 2019, 24, 3005 15 of 21 cathepsin could be directly measured by monitoring the degradation of the membranes. The low enzyme concentration could also be detected by increasing the crosslinking density of the peptide to increase the degradation rate (i.e., sensitivity). MoleculesA conductive2019, 24, x FOR hydrogel PEER REVIEW based on graphene oxide, dopamine, and polyacrylamide was prepared15 of 22 using the Schiff reaction, as shown in Figure 12[ 37]. The high stretchability, toughness, and adherenceself-adherence of the of conductive the conductive hydrogel hydrogel provided provided great greatbenefits benefits as a biosensor. as a biosensor. Wrist Wristpulses pulseswere successfullywere successfully detected detected through through instantaneous instantaneous changes changesin the electrical in the electricalcurrent and current resistance and resistanceusing the skinusing attachment the skin attachmenttest. Hydrogels test. had Hydrogels good self-absor had goodption self-absorption ability which was ability similar which to natural was similar tissues, to andnatural good tissues, cell andcompatibility, good cell compatibility,allowing them allowing to be them implanted to be implanted in vivo inwithout vivo without causing causing any inflammation.any inflammation. The conductive The conductive hydrogel hydrogel sensor sensorwas implanted was implanted into rabbits, into rabbits,and the andsignal the for signal muscle for fluctuationmuscle fluctuation was easily was easilyobtained, obtained, supporting supporting the potential the potential for forclinical clinical applications applications (the (the animal animal experimentsexperiments were were performed performed in in accordance accordance with with protocols protocols approved approved by by the the local local ethical ethical committee committee andand laboratory laboratory animal animal administration administration rules rules of of China). China). FigureFigure 12. 12. ConductiveConductive hydrogel hydrogel as as self-a self-adhesivedhesive electrode electrode biosensors. biosensors. (A1 (A1) )The The hydrogel hydrogel self-adhered self-adhered toto humanhuman arm arm skin skin and detectedand detected the electromyographic the electromyographic signals. (A2 )signals. Comparisons (A2) of Comparisons electromyography of electromyographyrecorded by hydrogel recorded electrodes by hy anddrogel commercial electrodes electrodes and commercial after ten electrodes cycles of usage after (adhere-strip).ten cycles of usage(B) The (adhere-strip). schematic diagram (B) The of schematic the chemical diagram structure of the of chemical conductive structure hydrogels. of conductive (C) Three hydrogels. hydrogel (electrodesC) Three hydrogel were implanted electrodes onto were the implanted dorsal muscle, onto the and dorsal the muscle, wires from and thethe electrodeswires from werethe electrodestranscutaneously were transcutaneously connected to theconnected signal to detector. the signal (D detector.) Example (D) of Example electromyography of electromyography recorded recordedby the implanted by the implanted hydrogel hydrogel electrode electrode biosensors biosensors from the from muscle the when muscle the when target the rabbit target was rabbit exposed was exposedto external to external stimuli (the stimuli animal (the experiments animal experiments were performed were performed in accordance in accordance with protocols with protocols approved approvedby the local by ethical the local committee ethical andcommittee laboratory and animal labora administrationtory animal administration rules of China). rules Reprinted of China). with Reprintedpermission with [37 ].permission© 2016, Wiley-VCH [37]. © 2016, (Weinheim, Wiley-VCH Germany). (Weinheim, Germany). AA hydrogel hydrogel membrane membrane assembled assembled layer layer by by layer layer from from partially partially aldehyde-dextran, aldehyde-dextran, chitosan, chitosan, and and glucoseglucose oxidaseoxidase (GOD)(GOD) was was reported reported for sensingfor sensing glucose glucose changes changes [105]. Since[105]. the Since GOD the in theGOD membrane in the membraneconverted theconverted external the glucose external into glucose gluconic into acid, gluconic which acid, reduced which the reduced local pH the value local of pH the value membrane of the membraneand triggered and the triggered expansion the ofexpansion the pH-sensitive of the pH membrane.-sensitive membrane. Fabry–Perot Fa fringesbry–Perot were fringes induced were by inducedthe swelling by the of swelling the hydrogel of the membranehydrogel membrane caused by caused glucose, by whichglucose, showed which showed the change the ofchange glucose of glucoseconcentration. concentration. GOD operated GOD operated well under well physiological under physiological pH conditions, pH conditions, exhibiting highexhibiting anti-interference, high anti- interference,and no response and no to response potential to interferences. potential interfer Particularly,ences. Particularly, the hydrogel the sensinghydrogel membrane sensing membrane showed a showedlinear response a linear withinresponse the within clinically the clinically relevant glucoserelevant range,glucose and range, the responseand the response speed was speed also was fast. alsoThe fast. resultant The resultant glucose biosensor glucose biosensor may have may the potential have thefor potential real-time for and real-time continuous and continuous glucose monitoring. glucose monitoring. 4. Summary and Outlook Hydrogels constructed using dynamic Schiff base linkages have been widely used in the biomedical field because of their non-stimulated uncoupling and recoupling under mild conditions. In this review, we focused on the categories, chemical mechanisms, and differences between the various types of Schiff base linkages, and we introduced the applications of hydrogels formed by the Molecules 2019, 24, 3005 16 of 21 4. Summary and Outlook Hydrogels constructed using dynamic Schiff base linkages have been widely used in the biomedical field because of their non-stimulated uncoupling and recoupling under mild conditions. In this review, we focused on the categories, chemical mechanisms, and differences between the various types of Schiff base linkages, and we introduced the applications of hydrogels formed by the Schiff base in the biomedical area, including drug delivery, wound healing, tissue regeneration, bioprinting, biosensors, and tissue adhesives. Various types of Schiff base linkages have applied in hydrogel formations. Imine, hydrazone, acylhydrazone, or oxime are formed through reactions between primary amine, hydrazine, acylhydrazine, or aminooxy and aldehyde. Amongst imines and their derivatives, acylhydrazones exhibit the best hydrolytic stability. Moreover, the bond stability of imines can be improved by connecting the benzene rings to the carbon or nitrogen atoms of the carbon–nitrogen double bonds. Therefore, Schiff base formations between benzoic aldehydes and acylhydrazines have gained significant attention in recent researches. Primary amine groups exist across a number of natural polymers, while chemical functionalizations are usually necessary to introduce the aldehyde groups or nucleophilic groups to the polymers. Periodate-mediated oxidation of polysaccharides is the most common method to introduce aldehyde groups through the cleavage of vicinal diols. Hydrazide and aminooxy groups can be conjugated with polymers through chemical modifications for hydrazone and oxime formations, respectively. Such hydrogels based on the Schiff reaction have self-healing ability, injectability, and other smart properties, owing to the reversibility and rapidity of the Schiff reaction. The Schiff reaction naturally exists in the human body and it plays an important role in the self-healing ability of human tissues. Citing strain sensitivity, biocompatibility, and self-healing properties, hydrogels composed of Schiff base linkages can be injected in situ and can integrate as a whole to display their various functions in minimally invasive and precise medical treatments, without the limitation of location or shape. Meanwhile, the aldehyde groups in the hydrogel can also bind to some integrins containing amine groups in human tissues, which can better combine with the host tissues. All the above properties show that smart hydrogels have great potential for applications in the biomedical field. Understanding the chemical mechanisms of different Schiff base linkages and the crosslinking process of hydrogels is of great significance to the design of smart hydrogels for biomedical applications. Hydrogels based on the Schiff reaction with universal self-healing properties have good biocompatibility, injectability, and easy functionalization, which has effective applications in drug delivery, bioprinting, tissue adhesives, and wound healing. Meanwhile, several research teams are gradually introducing innovative elements into the dynamic hydrogel network, and they are developing multi-component smart hydrogels with self-healing properties, conductivity, magnetism, optical, or thermal effects. Such smart hydrogel systems will have wide applications and prospective uses in cancer treatment, tissue regeneration, medical imaging, soft actuators, or artificial organs. Author Contributions: J.X. and Y.L. wrote the manuscript in consultation with S.-h.H. 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