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Page 1 of 44 New Journal of Chemistry
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1 The chitosan hydrogels: From structure to function

2
3 Jing Fu 1,2, Fuchao Yang 1, Zhiguang Guo1,3*
New Journal of Chemistry Accepted Manuscript

1
4 Hubei Collaborative Innovation Centre for Advanced Organic Chemical Materials and Ministry
Published on 17 September 2018. Downloaded by Freie Universitaet Berlin on 9/21/2018 8:43:25 AM.
5 of Education Key Laboratory for the Green Preparation and Application of Functional Materials,
6 Hubei University, Wuhan 430062, P. R. China.
2
7 School of Chemistry and Environment Engineering, Wuhan Institute of Technology, Wuhan
8 430205, P. R. China.
3
9 State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese
10 Academy of Sciences, Lanzhou 730000, P. R. China.
11 *Address correspondence to E-mail: [email protected]
12 Abstract
13 Knowledge of the structure of the hydrogels and the gelation mechanism of intelligent
14 hydrogels is essential to designing bioinspired hydrogels. As one of the raw materials
15 in hydrogels, chitosan has been highly pursued due to the polymer's biocompatibility,
16 biodegradability and low toxicity. Following the biomimetic principles to prepare
17 chitosan intelligent hydrogel has drawn great research interests in materials science
18 and engineering. In this paper, we present the recent progress in fabricating intelligent
19 chitosan hydrogels. Starting from the features of the hydrogels and the advantage of
20 the chitosan, the several development of the chitosan intelligent hydrogels have been
21 introduced. With constructed the hydrogels network, diversified physical interactions
22 and chemical covalent bonds have been used, this review focuses on the interactions
23 formed the hydrogels which based on the chitosan. The stimuli responsive
24 characteristics, bio-inspired functions and high mechanical strength properties of the
25 chitosan hydrogels have been detailed in the review. The emphasis of this paper was
26 on the relationship between the structural characters and the corresponding functions
27 of above intelligent hydrogels. Finally, a perspective on challenges facing the field
28 and prospects for future development are given.
29 Keywords: Chitosan; Hydrogels; Formation; Intelligent
30 1. Introduction
31 Hydrogel is a kind of hydrophilic polymer network which can swell but not dissolve
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32 in water, and it has character like both liquid and solid [1]. Despite the water in the
33 hydrogel is bound with the gel network, it is still a certain activity. The fully swollen
34 hydrogel shows some characteristic properties similar to the organism, including a

35 soft and rubbery consistency common to living tissues, higher permeability to
36 undersized molecules and release of entrapped molecules in a controlled manner [2-4].

37 In addition, the hydrogel can be prepared with many different physical forms, from
38 solid molded forms to membranes or sheets, and even to microparticles or coatings [5].
39 The dimensions of hydrogel can also vary from nanometers to centimeters in width,
40 and it is also relatively deformable and readily conform to the shape of any confined
41 space [6,7]. These features have made hydrogel applied on biomedical, medicine,
42 industry, agriculture and other fields [8]. Recently, new ideas on the design of
43 hydrogels with the response to stimulate of external environment [9,10] (such as
44 temperature, pH value, electricity, light, magnetic field, specific biological molecules,
45 etc.), self-repair characteristic like the organism and substantially enhanced
46 mechanical properties have broadened their applicability, these novel hydrogels which
47 different with the traditional hydrogels can termed intelligent hydrogels [11,12]. As
48 the first biomaterials designed for use in the human body, intelligent hydrogels have
49 revitalized biomaterials research field [13].
50 Comparing with the synthetic polymer hydrogels, the natural polymer hydrogels
51 have acquired increasing attention due to their properties of low toxicity, good
52 biocompatibility, easy degradation, rich source and low price [14]. Chitin is a natural
53 biopolymer with the most abundant polymer after cellulose. Chitosan (CS) derived
54 from deacetylated chitin (Fig. 1), is a natural amino homogeneous linear
55 polysaccharide composed of glucosamine and N-acetyl glucosamine units linked by
56 β-(1-4) glycosidic bond [15]. This material with free amino groups has no pollution to
57 the environment, owning to stronger reactivity and solubility than chitin [16]. Chitin
58 and chitosan can be easily processed into hydrogel [17,18]. Madhumathi et al.
59 synthesized chitin/nanosilver composite scaffolds with incorporation silver
60 nanoparticles into chitin hydrogel, and the scaffolds will function as ideal wound
61 dressings [19]. Jayakumar group developed α- and β-chitin membranes using α- and
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62 β-chitin hydrogels [20]. The bioactivity and cell adhesion studies of these membranes
63 indicated that it could be used for tissue-engineering applications. Chitin hydrogel is
64 usually prepared through a two-step process involving dissolution followed by

65 cross-linking, and specific solvent systems are required to dissolve chitin, which
66 possess poor solubility characteristic due to the numerous inter- and intra-molecular
67 hydrogen bonds between polymeric chains. Only a few solvents have been used for
68 the dissolution of native chitin, such as lithium chloride/dimethylacetamide [21],
69 calcium chloride dihydrate/methanol [22] etc. However, these polar solvents has
70 somewhat alleviated the issues with the biopolymer’s intractability, and the toxicity or
71 corrosivity of these organic components can inhibit batch production and potential
72 applications of the resulting gels. In recent years, people began to use chitosan as raw
73 material to prepare intelligent hydrogels and achieved some interesting results.
74
75 Fig. 1 Chemical structure of chitin and chitosan. Reproduced from ref. 15 with permission from
76 Elsevier, copyright 2004.
77 Today, the chitosan intelligent hydrogels have been widely used in separation and
78 cell culture, tissue engineering, enzyme immobilization, drug-controlled release and
79 targeted drug etc. [23,24]. For this purpose, many new strategies were applied in
80 fabricating hydrogels, such as Layer-by-Layer (LbL) assembly, soft-nanoimprint and
81 electrodeposition [26-28], and various structure with desirable functionalities chitosan
82 hydrogels have been developed. Ladet et al. developed a complex chitosan hydrogels
83 with multi-membrane ‘onion-like’ architectures which formation force were hydrogen
84 bonds, hydrophobic interactions and chitosan crystallites [29]. This physical hydrogel
85 could be used to encapsulate drugs for the co-delivery of multiple therapeutics or
86 pulse-like delivery of a given payload [30]. Encapsulation of chondrocytes and the
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87 delivery of biological therapeutics with thermo-setting chitosan hydrogel was reported

88 by Chenite's group [31], who designed thermally sensitive chitosan/glucose-phosphate
89 salts system which formed monolithic gels at body temperature, several driving forces

90 including hydrogen bonding, electrostatic interactions and hydrophobic interactions
91 were contributed to gel formation. Wei et al. fabricated a novel chitosan-based

92 hydrogel with the excellent self-healing property and good cytocompatibility[32]. The
93 self-healing efficiency of the hydrogel can achieve 95% under physiological
94 conditions, healing mechanisms of gel based on dynamic formation of carbon–
95 nitrogen bonds. It is worth noting that these amazing properties or smart behaviors of
96 chitosan intelligent hydrogels largely result from the interactions between molecules
97 which construct the polymeric networks and the cross-linking mechanisms of the
98 hydrogel formation.
99 Presently, many previous review articles have discussed the research advances of
100 chitosan hydrogel with structure or applications [19,20,33], however, few review
101 articles combine the structure with the smart behaviors to be considered.
102 Understanding the interactions formed the hydrogels and the mechanism of various
103 intelligent chitosan gels is valuable for the designing of multi-functional chitosan
104 hydrogel to adapt to wide applications. In this article, the recent advances of chitosan
105 hydrogels from their structures to functions were reviewed, especially in latest decade
106 years. Firstly, the approach of designing chitosan gels with the physical interactions or
107 chemical reactions was detailed analysis. Next, the mechanism of chitosan based
108 environment-sensitive hydrogels, the chitosan self-healing hydrogel and adhesive
109 chitosan hydrogel which bioinspired from the organism, and several type of high
110 strength hydrogel with chitosan based were summarized. At last, we discuss the
111 current challenges and conclude with the future perspectives for chitosan intelligent
112 gels. The intention of this mini-review is to give a brief critical overview of the recent
113 literature introduce the interactions among the hydrogel networks and explain the
114 reason with various intelligent behavior to suggest some potential design strategies for
115 fabricating multi-functional chitosan gels.
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116 2. Formation of the chitosan hydrogels

117 Chitosan based hydrogels are interconnected by various interactions into formation of
118 a 3D network. At present, the most reasonable classification method according to the

119 fabrication of gel network of hydrogels is the dominant interaction in the crosslinked
120 structure whether is covalent links [33]. Physical hydrogels are formed by secondary
121 interactions (Fig. 2a), such as electrostatic attraction, hydrogen bonding, hydrophobic
122 interaction and crystallization. Covalent bonds which be constitute of complementary
123 chemical groups are the main interactions to form the networks to the chemical
124 hydrogels. Crosslinker is added to react with chitosan for forming chemical hydrogels.
125 Certainly, in each of the two types of structures, physical interactions (such as
126 hydrogen bridges and hydrophobic interactions etc.) always participate in the
127 formation of the hydrogel more or less, but the standard of this classification to the
128 hydrogel is whether physical interactions or covalent bonds is the main interactions to
129 form the networks.
130 2.1 Physical hydrogels

131 2.1.1 Electrostatic interactions
132 Chitosan is a cationic polysaccharide derived from the natural polymer, electrostatic
133 interaction can occur between chitosan and negatively charged molecules and anions
134 because of the cationic amino groups of chitosan. Electrostatic interaction plays a
135 dominant role in the formation of chitosan physical hydrogels.
136 Many low molecular weight ions such as sulfates [34], citrates [35], and phosphates
137 [36] can mixed with chitosan to form gels [37], the use of low molecular weight ions
138 to prepare an ionic cross-linking polymeric matrix was found to be very simple and
139 mild. These ionic cross-linking hydrogels can vary material properties through the
140 interactions which depend on the concentration and degree of deacetylation with the
141 chitosan, as well as the size and charge density of the anionic agents [38,39]. Jing et al.
142 introduced ureido group into chitosan to get ureido-conjugated chitosan (UCCs) and
143 prepared the ureido-conjugated chitosan (UCCs)/sodium tripolyphosphate (TPP)
144 nanoparticles with magnetic stirring at room temperature using the ionic gelation
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145 method [40]. The formation of nanoparticles hydrogel was attributed to electrostatic
146 interaction between free amino group of chitosan and TPP. The characteristic study
147 indicated that UCCs/TPP hydrogel showed pH-sensitive performance and can be used

148 as a promising oral drug loading system targeted H. pylori. Introduced nanoparticles
149 (such as ZnO, Ag etc.) into chitosan hydrogels to generate organic-inorganic

150 nanocomposite hydrogels are found proper to be used as drug delivery carriers
151 because they frequently, exhibit remarkably improved properties compared with the
152 pure chitosan hydrogels [41,42]. Yadollahi et al. successfully prepared physically
153 cross-linked nanocomposite chitosan hydrogel beads using sodium tripolyphosphate
154 as the cross-linker [43]. The chitosan/silver nanocomposite hydrogel (CH/AgNPs)
155 was synthetized in situ formation of AgNPs in the chitosan hydrogel matrix. The
156 silver nanoparticles caused the increased antimicrobial activity and drug release
157 ability. With the same method, Yadollahi team incorporated ZnO nanoparticles into
158 chitosan matrix [44]. The prepared chitosan/ZnO nanocomposite hydrogels revealed a
159 higher swelling capacity and excellent drug release properties. Based on these
160 findings, the chitosan/functional nanocomposite hydrogels could be hopeful
161 candidates for the controlled delivery of drugs.
162 Polyelectrolyte complexed (PEC) hydrogels are formed by electrostatic interactions
163 between chitosan and anionic polymers. The difference between the ionic
164 cross-linking hydrogels and PEC is the molecules to from the PEC with a broad MW
165 range [45]. Most of anionic of polysaccharide (e.g. Alginate [46], k-Carrageenan [47],
166 Chondroitin sulfate [48], Dextran sulfate [49], Gellan gum [50], Pectin [51], Xanthane
167 [52], Xylan [53]), protein (e.g. Silk fibroin protein[54], Collagen [55], quinoa protein
168 [56]) and synthetic polymer (e.g. PAA [57], Polyphosphate [58], Poly (L-lactide) [59])
169 can be formed PEC with chitosan. In the process of preparation PEC, no auxiliary
170 molecules are needed to add in the reaction system, besides chitosan and polyanionic
171 polymer. Therefore, it is favourite advantages due to avoid complicated purification
172 operations and reserve biocompatibility of polymers. The characteristics of
173 chitosan-based PEC can be changed by the chemical characteristics of the polymers’
174 components, such as the molecular weight (MW), charge density, functional group
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175 structure, hydrophobicity/hydrophilicity balance, stereo-regularity, flexibility, and

176 compatibility, as well as the reaction conditions (including solvent, ionic strength, pH,
177 and temperature) [60]. PEC formation reaction have to occur at ionized polymers with

178 opposite charges which means the pH value of the complexation only be chosen in the
179 vicinity of the pKa interval of the two polymers [61]. Conzatti et al. prepared PEC
180 based on alginate and chitosan for biomedical applications, and they studied the effect
181 of the drying process on PEC [62]. The results indicated different type structures of
182 PEC with different porosity scale depend on the choice of the drying technique, and it
183 can be evaluated as potential internal wound dressings.
184 2.1.2 Hydrogen bonding and hydrophobic interactions
185 In 2000, Chenite and co-workers first reported a chitosan-based thermogelling
186 systems which be made of chitosan and glycerophosphate (GP) with injectable,
187 neutral and thermally sensitive properties [31]. Many recent studies proposed
188 explaining the CS/GP system gelation mechanism, the reasonable explanation is that:
189 i) in lower temperature, with the reduced electrostatic repulsion force in chitosan
190 interchains, hydrogen bonding between multi-hydroxyls in the chitosan chain and
191 water play a key role to avoid the polymer chain twined each other, ii) when the
192 temperature increased, the hydrophobic interactions among chitosan chains presumed
193 to be the main driving force of the gelation mechanism with the hydrogen bonding
194 broken [63,64]. The particular temperature that the polymer solution undergoes
195 solidification into hydrogel was defined as the lower critical solution temperature
196 (LCST) [65]. This means that the hydrogel take advantage of hydrophobic
197 interactions to form network between chains above the LCST. Recently, other
198 chitosan-based hydrogel systems were developed using this mechanism to yield a
199 semi-rigid gel from a flowable liquid solution. Bhattarai et al. engineered
200 chitosan-PEG hydrogel [66] that utilized the hydrophobic interactions between the
201 polymer chains to form junctions above the transition temperature [67,68]. Chen et al.
202 obtained chitosan- poly(N-isopropyl acrylamide) (PNiPAM) thermoreversible gel [69].
203 It is believed that at low temperature the hydrogen bonding between polymer polar
204 groups and water molecules result in polymer dissolution, while at high temperature
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205 the hydrophobic interactions between the polymer chains dominate. These hydrogel
206 systems will play a very significant role in the biomedical and pharmaceutical
207 applications [70,71].

208 2.1.3 Multi-physical interactions
209 Some hydrogels can be formed not with specific physical interactions but with
210 multi-physical interactions, such as the biocompatible chitosan/PVA complex [72].
211 The structure of chitosan/PVA hydrogel can be considered as directly interact together
212 via hydrogen bonds and crystallization [73]. The hydrogel structure with slightly
213 difference depended on the preparing method that was used (Fig. 2b). Hydrogen
214 bonds between hydroxyl groups of PVA and hydroxyl or amino groups of chitosan
215 were the main interactions inside the network formed by the autoclaving method [74].
216 While in addition to these interactions, crystallite junction zones between polymeric
217 chains and interpolymer complexation were form junction points with lyophilization
218 or freeze–thaw method [75]. The structure and properties of chitosan/PVA hydrogel
219 can be influenced by the ratio of chitosan/PVA. Khan et al. prepared
220 chitosan/polyethylenimine (PEI) hydrogel [6]. It is assumed that the gel network was
221 linked together by chitosan–chitosan interactions. Ladet et al. reported a
222 multi-membrane onion-like physical hydrogel which formed neutralization in a
223 sodium hydroxide solution and subsequent washings alcohol gel in water [29]. The
224 formation force of this physical hydrogel can be considered to hydrogen bonds,
225 hydrophobic interactions, and chitosan crystallites rather than ionic repulsion. These
226 hydrogels certainly represent a better medium to encapsulate drugs for the co-delivery
227 of multiple therapeutics than covalently crosslinked hydrogels due to no auxiliary
228 molecules or crosslinkers were required [30,76].
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229
230 Fig. 2 Physical hydrogels a) Schematic representation of chitosan-based hydrogel networks
231 derived from different physical associations. Reproduced from ref. 45 with permission from
232 Elsevier, copyright 2010. b) Structure of a chitosan/poly (vinyl alcohol) (PVA) complex hydrogel;
233 (left) prepared by the autoclaving method; (right) prepared by the freeze–thaw method.
234 Reproduced from ref. 33 with permission from Elsevier, copyright 2004.
235 Physical hydrogels are networks held together by molecular entanglement and/or
236 secondary molecular interactions. Since the assembly of physical hydrogels depend
237 on the spontaneous organization and specific association of molecules through a
238 number of non-covalent interactions, physical hydrogels can also be called
239 “self-assembling hydrogels” [77,78]. Some of the physically cross-linked hydrogels
240 exhibit viscous flow under shear stress (shear-thinning) [79]. Shear-thinning
241 behaviour enables a pre-formed hydrogel with desired physical properties, as
242 characterized ex vivo, to be delivered in vivo via application of shear stress during
243 injection (most commonly by passing through a syringe). Self-assembly is the main
244 route for cross-linking for shear-thinning hydrogels. Self-assembly is obtained as a
245 result of a balance between competing forces that favor assembly (e.g., hydrophobic
246 interactions, hydrogen bonding and electrostatic attraction) and forces that act against
247 assembly (e.g., electrostatic repulsion and solvation) [80]. Due to the dynamic nature
248 of these weak physical associations, formed networks can be dissociated under
249 applied shear. Shear-thinning is one promising technique for the application of
250 injectable hydrogels, and these gels can be used to deliver biological molecules and
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251 cells during the injection process.
252 2.2 Chemical hydrogels

253 Compared with physical chitosan hydrogels, the covalent cross-linking networks of

254 chitosan based are easier to be controlled due to some interesting properties of the
255 hydrogel such as mechanical strength and drug release can modulate with the
256 crosslinking density of crosslinking reaction. The cross-linking density can be
257 influenced by many parameters: i) cross-linking agent types; ii) cross-linked agent
258 concentration; iii) the number or activity of crosslinked sites; iv) cross-linking
259 methods (surface cross-linking or bulk cross-link). Crosslinkers are the agent with one
260 or more reactive functional groups that allow the formation of covalent bonds
261 between polymeric chains. The common stategy divided chemical chitosan hydrogels
262 (or covalently crosslinked chitosan hydrogels) based on the crosslinkers, but the main
263 drawback of this classification method is that it can’t contain all of the crosslinkers. In
264 this section, we tried to divide the chemical chitosan gels from the perspective of
265 chemical bonds (Table 1). Chemical modification to introduce a variety of functional
266 groups into chitosan chains was a popular way to design the chitosan based hydrogels.
267 However, with respect to chemical chitosan hydrogels, it is not entirely suitable if 3D
268 network formed with the introduced functional groups rather than covalent bonds
269 were built between functional groups in the chitosan and crosslinkers. For this reason,
270 we only detailed the covalent bonds which originated from the chitosan chains
271 directly.
272 2.2.1 Imine bond (Schiff base)
273 Imine bond (also known as imine, ―N=CH―), commonly called Schiff base, is the
274 most frequently used as covalent bond for creating chitosan-based hydrogels. The
275 aldehyde groups can form covalent imine bonds with the amino groups of chitosan.
276 Dialdehydes (in particular glutaraldehyde (GA)) were the most commonly used
277 cross-linkers with chitosan-based biomaterials [81]. The cross-linking mechanism was
278 studied by Webster [82]. In fact, there are many advantages crosslinked GA with the
279 chitosan, such as the easiness of synthesis, the speed of reaction, the mild
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280 experimental conditions, the acceptable biocompatibility of the reactants, but the main
281 challenge is how to remove the excess neurotoxic glutaraldehyde.
282 Monoaldehydes – which are widespread in nature, cheap and beneficial to the

283 human body, were applied in the crosslinking agent when prepared the chitosan
284 hydrogels. Iftime et al. revealed a method of obtaining chitosan hydrogels using
285 monoaldehydes [83]. Hydrogels based on chitosan and salicyladehyde were obtained
286 by forming of the covalent imine link-age. The hydrogels were thermosensitive,
287 exhibited self-healing properties and demonstrated good mechanical properties. Singh
288 et al. reported the preparation of chitosan hydrogels by crosslinking with
289 formaldehyde used as crosslinking agent [84]. The network is easily established via
290 Schiff base between chitosan and formaldehyde, and exhibited a typical pH and
291 temperature responsive behavior.
292 Imine bond is produced from the aldehydes not only intrinsic functions but also
293 introducing aldehyde via oxidation reactions. The vicinal glycols in many saccharides
294 and polysaccharides can be cleaved with periodate oxidation to form their dialdehyde
295 derivatives, introducing aldehyde functions which serve as crosslinkers for polymers
296 with chitosan’s primary amino groups to form hydrogels [85]. Yao and coworkers
297 synthesized alkylated chitosan gels via Schiff base formation by oxidized glucose
298 cross-linked chitosan chain [86], and the membrane with non-toxic crosslinker
299 oxidized glucose can be applied in drug delivery. Weng et al. obtained Odex/ CEC
300 hydrogel composed of oxidized dextran (Odex) and N-carboxyethyl chitosan (CEC).
301 This system could gel at body temperature rapidly via Schiff base formation between
302 the free ―NH2 groups on the N-carboxyethyl chitosan and ―CHO functionalities of
303 the Odex [87]. The investigations implied the gelation time (30-600s) could be
304 modulated by changing the concentration or the ratio of Odex/CEC to suitable for
305 designing in situ injectable systems. Li groups have successfully prepared a versatile,
306 non-toxic, in situ hydrogel based on N,O-carboxymethyl chitosan and oxidized
307 alginate without using additional crosslinking agents [88]. The rapid sol–gel transition
308 of N,O-carboxymethyl chitosan/oxidized alginate hydrogel also occurred at
309 physiological temperature, suggesting that this system might serve as an injectable in
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310 situ gelling system for local drug delivery system. Li et al. oxidized hyaluronic acid
311 (A-HA) to generated reactive aldehyde groups and introduced carboxymethyl groups
312 to O-position of the glucosamine units of chitosan (NOCC) [89]. The aldehyde groups

313 in A-HA could cross-link with amino groups in NOCC via Schiff’s base linkage to
314 obtain NOCC/A-HA hydrogel. The hydrogel was effective in reducing the formation
315 of intraperitoneal adhesions after surgery. Chetouani et al. used oxidized pectin as
316 crosslink agent to form hydrogel [90]. Cross-linking reactions between chitosan and
317 oxidized pectin have been evidenced, avoiding the use of toxic crosslinking reagents.
318 The use of this type of films improved the antibacterial activity of chitosan, which
319 demonstrates them to be promising materials for biomedical applications, such as
320 wound dressing. In addition, galactomannan, maltodextrins and methylcellulose [91],
321 xyloglucan [92], and scleroglucan [93] can all be oxidized to obtain dialdehyde
322 structure and reacted with amino groups to form hydrogels.
323 Other strategy to synthetize imine bond hydrogels is grafted small molecular
324 aldehyde in the end of polymer to crosslink with chitosan. Zhang et al. prepared a
325 multi-responsive, dynamic, and self-healing chitosan-based hydrogels [94]. In their
326 work, a dibenzaldehyde-terminated telechelic poly(ethylene glycol) (PEG) was
327 synthesized and was allowed to form Schiff base linkages between the aldehyde
328 groups and the amino groups in chitosan. The hydrogels were found to be
329 self-healable and sensitive to many biochemical-stimuli, such as pH, amino acids, and
330 vitamin B6 derivatives.
331 2.2.2 Amide bond
332 The amide bond is one of covalent bond based on esterifying agents including
333 carboxylic acids or carboxylic acid derivatives reacted with amino groups. Tsao et al.
334 developed the Methoxy-poly (ethylene glycol)/ chitosan (mPEG-g-chitosan) hydrogel
335 with multi-stimuli sensitivity [95], in which the amide linkages formed between
336 chitosan and mPEG-acid to result in yielding mPEG-g-chitosan. Araki et al. prepared
337 nanocomposite gels by hexamethylene-1,6-di-(aminocarboxysulfonate) (HDS)
338 mediated cross-linking of chitosan [96], and the cross-link density may affected the
339 degree of swelling and the mechanical properties of the nanocomposite gel. Zhang et
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340 al. utilized two kinds of polyelectrolytes with opposite charges to develop the
341 poly(L-glutamic acid)/chitosan scaffold [97]. A hydrophilic network created with
342 amide bonds cross-linked carboxyl groups on the PLGA into amine groups in the CS

343 chains. This hydrogel scaffold with the protein repulsive and porous was evaluated for
344 cartilage tissue engineering, and the result proved the material has biocompatibility
345 and degradation.
346 2.2.3 Heterocyclic amine and Secondary amine bond
347 Compared with other covalent cross-linkers, genipin(GN) which extract form
348 gardenia jasminoides ellis is desirable to use an alternative cross-linking reagent that
349 could lead to the formation of stable and biocompatible cross-linked products [98].
350 Different cross-linking mechanism originated from different sites on the genipin
351 molecule. Two step of crosslinked reactions was involved in the following: i) The
352 carboxymethyl group of genipin firstly reacts with amino group of chitosan to form
353 secondary amide; ii) nucleophilic attack by amino group of chitosan on the olefinic
354 carbon atom at C-3 of deoxyloganin aglycone followed by the opening of the
355 dihydropyran ring to form heterocyclic amine [99]. The crosslinked network
356 formation between the bifunctional linkages of genipin with amino group of chitosan
357 could further go through apolymerization to for moligomer-bridge. Mi et al. showed
358 that the signiﬁcant difference in swelling behavior of hydrogel exhibit sensitivity to
359 the environmental pH owing to the different crosslinking extents and different chain
360 lengths of crosslink bridges [100,101].
361 Etherifying agents including organochlorine, epoxide etc. can react with amino
362 radical in the chitosan chains to result in formation of secondary amine bonds (R–
363 NH–R). Subramanian et al. used 1,4-butanediol diglycidyl ether to crosslink the
364 chitosan chains via the reactive amino group [102], the film hydrogel was found to be
365 nontoxic to chondrocytes and more hydrophilic. Kiuchi and coworkers achieved
366 chitosan-based hydrogel through a traditional epoxy-amine reaction between
367 diepoxy-PEG and chitosan [103]. The swelling behavior of the film greatly relied on
368 the molecular weight of the crosslinker PEG-epoxy and weight percent of the
369 diepoxy-PEGs.
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371
370
Table 1. The covalent bonds and network structures formed between the chitosan chain and common crosslinkers
Covalent Chitosan or
Crosslinkers Structure 3D networks Ref.
bonds chitosan derivative
Gutaraldehyde Chitosan [82]
Salicyladehyde Chitosan [83]
Formaldehyde Chitosan [84]
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Imine bond
Oxidized glucose Alkylated Chitosan [86]
N-carboxyethyl
Oxidized dextran [87]
Chitosan
Oxidized sodium N-carboxyethyl

[88]
alginate Chitosan
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Page 15 of 44
372
Covalent Chitosan or chitosan
bonds derivative
Oxidized N,O-carboxymethyl
[89]
hyaluronic acid Chitosan
Oxidized pectin Chitosan [90]
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Oxidized
Imine bond Chitosan [91]
xyloglucan
Oxidized
Chitosan [93]
scleroglucan
Dibenzaldehyde-t
erminated
telechelic Chitosan [94]

poly(ethylene
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glycol) (PEG)

374
373
Covalent Chitosan or
bonds chitosan derivative
Methoxy-poly
Chitosan [95]
(ethylene glycol)
Hexamethylene-1,
6-di-(aminocarbo
Chitosan [96]
xysulfonate)
Amide bond
(HDS)
Poly(L-glutamic
Chitosan [97]
acid)
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Genipin Chitosan [99-101]
Heterocyclic
amine and
1,4-Butanediol
Secondary Chitosan [102]
diglycidyl ether
amine bond
Diepoxy-PEG Chitosan [103]
Chitosan chain or Chitosan derivative chain In the model, only a small number of polymer chains are depicted for simplicity.
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375 Chemical hydrogels have the advantage of precisely controlling the gel chemical
376 functionalization, degradation or dissolution and the characteristic correlated with the
377 adjusting structure. However, the crosslinker or the auxiliary molecules which used in

378 the functionalization process required performing an additional purification and
379 verification step before the administration of the hydrogel because of the toxic with
380 the above agents, this point is the main drawback of these systems. For the moment, it
381 is very important to choose the safe and biocompatible covalent crosslinkers.
382 In recent years, research interest has been focused on polymerized gels in situ, that
383 is, at the site of forming, without the aid of additional crosslinking agents. In situ
384 polymerized gels can be applied in many biomedical fields, including drug delivery,
385 cell encapsulation, and tissue repair. Due to the biocompatibility of chitosan, a variety
386 of physical and chemical crosslinking strategies were detailed previously and have
387 been used to fabricate in situ chitosan hydrogels [104].
388 3. Chitosan intelligent hydrogels

389 Some environment stimulation can lead to hydrogel system have dramatic changes in
390 their physical or chemical behavior, and this is the earliest known of intelligent
391 hydrogel for the researchers. Except stimuli-responsive hydrogels, self-healing
392 hydrogels can self-repair like the organism when it damaged and higher strength
393 hydrogels own the higher mechanical strength than the normal hydrogel, they can also
394 be classified as intelligent hydrogel.
395 3.1 Stimuli-responsive hydrogels

396 The stimuli-responsive hydrogel usually respond to environmental stimuli like living
397 organisms in nature. When environmental conditions (such as pH value, temperature,
398 electric or magnetic fields and light) altered, hydrogels are capable of undergoing a
399 discontinuous volume change and accompanied with the change of water content
400 (volume phase transitions). In essence, an infinitesimal change of the configuration
401 and size of polymer chains can be induced by the environmental intensive variable
402 (including pH value, temperature, ionic changes and so on). The changes of the
403 polymer chain motion trigger an enormous change of extensive properties (such as
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404 swelling behaviors, network structure, permeability, or mechanical strength) of

405 hydrogel, which can be regarded as volume phase transitions [105]. Therefore, the
406 hydrogel with responsive properties is of great importance in its application, as drug

407 delivery matrix, actuator, sensor, “on-off” valves and so on. These applications may
408 be limited by low responsive rate or low mechanical strength, so understanding of the
409 stimuli-responsive mechanism can lead to design the responsive rate controllable
410 chitosan-based hydrogels by changing the structure and composition of the chitosan
411 network or by changing the size of hydrogels.
412 3.1.1 Ion or pH Sensitive Gels
413 The characteristic phenomenon of ion or pH sensitive hydrogels is the dynamic
414 swelling corresponding to weakly acidic or basic pH changes in the surrounding
415 medium. General speaking, if the hydrogel polymer chain contains ionizable groups,
416 it becomes an ion or pH sensitive gel. When the pH and ionic strength varying, the
417 concentration difference of ion between the environment and hydrogel inside lead to
418 the hydrogel swelling or shrinkage, resulting in volume phase transitions (Fig. 3a).
419 Since chitosan is a polycationic copolymer with plenty of amino groups on the chain,
420 the pKa of chitosan [106] result in a strong response of chitosan hydrogel which has
421 sufficient free ―NH2 near or slightly acidic of neutral pH value with the ―NH2
422 combine or release proton. Physical cross-linking and chemical modification of
423 chitosan-based hydrogels have been developed for preparing ion or pH sensitive
424 hydrogels. The mechanism of pH sensitive attributed to the unreacted amino groups
425 which would be protonated in acid solutions or deprotonated in the basic solutions
426 into the chitosan or chitosan-derivative chains [107], so the hydrogels swelling and
427 pH sensitivity could be modulated with the components and the connection mode of
428 each component.
429 Wu et al. capped the porous SiO2 templates layer with crosslinked chitosan hybrid
430 hydrogel [108]. In this work, as a crosslinking agent, the
431 glycidoxypropyltrimethoxysilane (GPTMS) performs an effective crosslinking
432 function with cytocompatibile in vitro [109] for chitosan and react with the silanol
433 groups of the porous SiO2 surface via siloxy bonds [110]. Optical measurement of a
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434 sample was determined the thickness of chitosan/GPTMS hydrogel film when pH
435 changes from 7.4 to 6.0, thus, the reflectivity spectrum displayed this layer was very
436 sensitive to pH in this pH range. The concentration of GPTMS can adjust the response

437 time and swelling ratio of the hydrogel. They also investigated the pH-dependent drug
438 release properties of the insulin-loaded sample. The chitosan hybrid hydrogel layer on
439 the porous SiO2 surface forms an effective barrier and blocks insulin release in pH 7.4
440 solution, while the drug in the pores can continuously diffuse into solution from the
441 swollen hydrogel layer when the pH value is 6.0. This result indicated that the
442 hydrogel with the pH-dependent volume phase transition is an effective and
443 convenient strategy in drug delivery and biosensing via change in pH value.
444 3.1.2 Thermosensitive hydrogel
445 Chitosan based thermosensitive hydrogel is a type of intelligent gel which is
446 nonchemical crosslinked, it can manifest thermodynamic structural transitions along
447 with associated temperature change of external environment. When the
448 temperature-stimulated phase transition was be induced, the hydrogel may show as a
449 large change in the shape, rigidity, water content or hydrophobicity due to couple
450 water absorption and network deformation of the gel [111]. Generally, a change in
451 intermolecular interactions of the polymer chain in response to temperature can be
452 considered as the cause of thermosensitive behaviors in the hydrogel (Fig. 2b). The
453 polymer solution turn into a gel above the lower critical solution temperature (LCST)
454 [112], and LCST correlate with hydrophobic constituents of the polymer chain.
455 Therefore, the LUST of thermosensitive hydrogels can adjust the near physiological
456 temperature via grafting, block or blending some functional group or polymer into the
457 network [45]. Chitosan is not a thermosensitive polymer on its own, introducing
458 hydroxyl groups or some amphiphilic thermosensitive polymer into the chitosan
459 network to obtain thermosensitive hydrogels were usually strategies. For example,
460 grafted hydroxybutyl to chitosan [113], composited PEG [66] or PVA [114] into
461 hydrogels, blended polyol salt [115] into chitosan hydrogels belong to the front.
462 Pluronic [116] (triblock poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene
463 oxide), (PEO–PPO–PEO) copolymer), PNIPAAm [117]
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464 (poly(N-isopropylacrylamide)) or PNVCL [118] (poly(N-vinylcaprolactam)) are

465 amphiphilic thermosensitive polymer which can be mixed into chitosan hydrogels.
466 The hydrophilic–hydrophobic balance can be modulated with these functional groups

467 or polymers via temperature change, and varying the hydrophilic–hydrophobic ratio
468 in the hydrogels can adjust the thermosensitive capacity.

469 Chenite et al. added glycerophosphate (GP) to chitosan solution in an ice bath
470 under stirring, the mixed solution becomes thermoresponsive at physiological pH [31].
471 In this system, synergistic effect of hydrophobic interactions and hydrogen bonding in
472 the chitosan chains is the driving force for this gelation. Park et al. developed
473 thermosensitive chitosan Pluronic (CP) hydrogel as an injectable delivery carrier for
474 cartilage regeneration [116]. The CP hydrogel has a phase transition around body
475 temperature as a function of its Pluronic content [119].
476 3.1.3 Magnetic field sensitive hydrogel
477 Magnetic field sensitive hydrogel can control drug and cell release both in vitro and in
478 vivo with inducing volume phase transitions in an external magnetic field (MF) [120].
479 The magnetic-sensitive polymer is superior to the other stimuli response hydrogel (e.g.
480 pH or thermal sensitive gel) because MF is an action-at-distance force (noncontact
481 force) which can apply it in telemedicine treatment [121]. Since none of the soft
482 hydrogel has natural sensitive to MF, the magnetic hydrogels are obtained from
483 combination hydrogels with nanomagnetic particles (NMPs) (e.g. Fe3O4, γ-Fe2O3,
484 CoFe2O4 and ferrite etc.) [122,123]. Various methods for preparing magnetic
485 hydrogels have been developed, such as the grafting-onto method, a blending method
486 and an in situ precipitation method [124-126]. There are some advantages and
487 limitation in these fabrication methods, with the grafting-onto method, the uniformity
488 and stability of the NMPs dispersed within the hydrogel can be guaranteed since the
489 covalent bonds are formed between the NMPs and hydrogel network.
490 Chitosan may not be fit for fabricating magnetic hydrogels in grafted NMPs due to
491 its weak mechanical properties and the lack of active sites for MNPs. In contrast,
492 synthetic polymers (e.g., poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and
493 PAAm) may benefit the MNPs grafting onto hydrogel networks or in situ precipitation
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494 in hydrogels [127]. Therefore, Zhang et al. blended the magnetic Fe3O4 nanoparticles
495 which modified with carboxyl on the particles surface into chitosan solution to form a
496 stable ferrofluid. In this system, the mangetic nanoparticles could be dispersed well in

497 solution due to the interaction between carboxyl groups on the Fe3O4 surface and
498 -NH2 on chitosan [128]. Then, they crosslinked the ferrofluid with telechelic
499 difunctional poly(ethylene glycol) (DF-PEG), and the 3D networks formed with -NH2
500 groups on polymeric chains and benzaldehyde groups at PEG ends via covalent
501 Schiff-base linkage(imine bond). In this work, the Fe3O4 nanoparticles were fixed in
502 the hydrogel networks and the magnetic hydrogel was obtained with the grafting-onto
503 method. Because the imine bond could be considered as the reversible dynamic
504 covalent, this hydrogel could self-mend itself automatically without additional stimuli.
505 The self-healing function and magnetic sensitivities were combined well in the
506 hydrogel, e.g. the broken hydrogel pieces could be massed under an external MF, and
507 they realigned an integral gel. The magnetic self-healing hydrogel could pass through
508 a narrow channel with changing its shape but still maintaining an integral appearance
509 in an external magnetic field (Fig. 3c). In summary, magnetic-sensitive hydrogel is a
510 potential materials application in controllable release of drug system and remote
511 therapy microdevice.
512 3.1.4 Electrosensitive hydrogel
513 Hydrogel can swell, shrink, or bend under electric current. This kind of intelligent
514 hydrogel is electrosensitive hydrogel. Hydrogel sensitive to electric stimulus is
515 usually made of polyelectrolytes [129]. Under an electric field, electrosensitive
516 hydrogel undergo deformation attribute to the voltage-induced migration of ions with
517 expansion of one side and contraction of the other. Chitosan also have the
518 electrosensitivity due to it is a polycation polysaccharide. Moreover, the ionized
519 groups can be introduced into chitosan chain via the multi- hydroxyl structure of
520 chitosan. Three factors would influence the electrosensitive capacity of
521 chitosan-based electro-responsive hydrogel: i) the pH value of the medium [130]; ii)
522 the ionic strength of the medium [131]; iii) the electric voltage of the external electric
523 field [132].
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524 Chitosan/polycation hydrogel and amphoteric chitosan/polyanion hydrogel were

525 the most studied electro-responsive hydrogel according to incorporate cationic or
526 anionic polymers into the chitosan network. Kim group has a lot of work in

527 chitosan/polycation hydrogel. They fabricated chitosan/polyaniline hydrogel [133],
528 chitosan/polyallylamine hydrogel [134], chitosan/poly (vinyl alcohol) hydrogel [135]

529 and chitosan/polyacrylonitrile hydrogel [136]. These chitosan/polycation hydrogels
530 can experience greater swelling pressure on the side near the cathode than on the side
531 near the anode and lead to bend toward the anode because of the protonation of amino
532 and other cations, and they showed good sensitivity with reversible properties under
533 the electric field stimulation. Liu et al. examined electroresponsive behavior of IPN
534 hydrogel composed of 2-hydroxypropyltrimethyl ammonium chloride chitosan
535 (HACC) and PVA [137]. The results indicated that the bending behavior can be
536 affected by the ionic strength of the electrolyte and the voltage of the applied electric
537 field. However, the blending capacity of chitosan/polycation hydrogels decreased in
538 basic medium, which limited its applications in different pH conditions. Amphoteric
539 chitosan/polyanion hydrogel incorporated anionic groups into the chitosan network
540 for purpose of regulating the electro-responsive behaviors. Kim and coworker
541 prepared chitosan/hyaluronic acid PEC hydrogels [132] and chitosan/poly
542 (hydroxyethylmethacrylate) hydrogels [138] to obtain the amphoteric
543 chitosan/polyanion hydrogel. Shang research team prepared a natural amphoteric
544 electric-sensitive hydrogel by solution blending of CS and carboxymethylcellulose
545 [139] (Fig. 3d). In these system, cationic group (-NH2) and anionic group (-COOH)
546 coexist in the network in order to expand the range of pH value on the
547 electro-response. The hydrogels bend toward the anode in acidic medium but bend
548 toward opposite direction in basic medium due to the osmotic pressure difference
549 induced by the motion of ions. Because the electric field is the contactless and
550 convenient stimulus from the point of view of signal control, chitosan-based
551 electric-sensitive hydrogels which can directly transform electrical energy into
552 mechanical work have many potential applications in artificial muscle, drug delivery
553 systems, bionic actuator and chemical valve [140].
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554
555 Fig. 3 Stimuli-Responsive hydrogel a) The pH-responsive swelling of chitosan-based hydrogels.
556 Reproduced from ref. 105 with permission from CRC Press, copyright 2011. b) Schematic
557 representation of thermosensitive networks of physical gelation driven by hydrophobic
558 interactions. Reproduced from ref. 45 with permission from Elsevier, copyright 2010. c) Hydrogel
559 passes through a narrow channel by shape transformation under the assistance of an external
560 magnetic field. Reproduced from ref. 128 with permission from the Royal Society of Chemistry,
561 copyright 2012. d) Schematic diagram for testing the bending behavior of hydrogels. Reproduced
562 from ref. 105 with permission from the American Chemical Society, copyright 2008.
563 3.2 Bio-inspired hydrogels

564 3.2.1 Self-healing hydrogel
565 Self-healing is considered as one of the basic properties of living tissues, and the
566 artificial materials do not have this ability. However, an increasing number of
567 man-made hydrogels possess the self-repair properties when they are damaged [141].
568 Generally, two approaches are explored to fabricate self-healing hydrogel:
569 noncovalent reactions (physical crosslinking) and dynamic covalent reactions
570 (chemical crosslinking) [142]. Noncovalent interactions are normally utilized to
571 construct physical self-healing gels, including hydrophobic interactions, host–guest
572 interactions, hydrogen bonds, and crystallization etc. [143]. Chemical self-healing
573 hydrogel re-establish with dynamic covalent reactions, such as sulfur–sulfur bonds
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574 (disulfide), boron–oxygen bonds (phenylboronate ester), carbon–nitrogen bonds

575 (imine, acylhydrazone) and cyclohexenes (reversible Diels–Alder cycloaddition)
576 [144]. These reversible interactions or dynamic bonds can reform the networks to

577 restore their original structure and properties once the self-healing hydrogels were
578 broken (Fig. 4a). The hydrogels with self-repair ability contain at least separate or
579 combined interactions above-mentioned, so it is very important in designing the
580 special groups on the macromolecule and crosslinking between the polymer chains to
581 form the gels. Self-healing hydrogel is particularly promising for a variety of medical
582 applications owing to its biocompatibility and mechanical similarity to natural tissues
583 [145]. As injectable biomaterials, self-healing hydrogel has unique advantages in
584 minimize the risks and complications associated with surgical implantation [146].
585 Wei research group achieved a chitosan-based self-healing hydrogel by dynamic
586 covalent chemistry (Schiff-base linkage) to repair the central nervous system [147].
587 The gel was formed by crosslinking benzaldehydes at both ends of difunctionalized
588 PEG (DF-PEG) with glycol chitosan. Changing the concentrations of the polymer
589 and/or the crosslinker can adjust the stiffness of the gels. The self-healing capability
590 of the hydrogel was given by the cleavage and regeneration of the imine linkages in
591 the 3D networks. The visual observation and the rheological measurements revealed
592 that the chitosan-based self-mending hydrogel could recover its mechanical properties
593 and structure after high strain-induced structural damage (Fig. 4b). Drug delivery
594 experiments on animals suggested this self-healing gel exhibits a promising strategy
595 in repairing the central nervous system.
596 Chen group designed a chitosan-based hydrogel (CEC/OSA/ADH), which formed
597 by adding oxidized sodium alginate (OSA) solution into the mixture of
598 N-carboxyethyl chitosan (CEC) and adipic acid dihydrazide (ADH) [32]. In this
599 hydrogel, the network were constructed with two kinds of dynamic covalent bond:
600 imine bonds and acylhydrazone bonds (Fig. 4c). Imine bonds obtained from the
601 reaction of the amino groups (from CEC) with aldehyde groups (from OSA) and
602 acylhydrazone bonds produced by the condensation reaction between OSA and ADH
603 (the cross-linker) [148]. The dynamic covalent bonds impart the self-healing
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604 capability to the CEC/OSA/ADH hydrogel under physiological conditions. This

605 hydrogel can be used as injectable hydrogels for drug delivery and cell therapy.

606
607 Fig. 4 Self-healing hydrogel a) Schematic illustration of self-healing mechanisms of self-healing
608 gels. Reproduced from ref. 124 with permission from the Royal Society of Chemistry, copyright
609 2014. b) Gross appearance of the damaged and healed chitosan-based hydrogels. Reproduced from
610 ref. 147 with permission from Wiley-VCH, copyright 2015. c) Chemical structures and
611 photographs of self-healing process of the CEC/OSA/ADH self-healing hydrogel. Reproduced
612 from ref. 32 with permission from Wiley-VCH, copyright 2015.
613 3.2.2 Adhesive hydrogel

614 Tissue adhesive materials have attracted much attention as promising materials
615 applicable for wound healing patches, tissue sealants, and hemostatic materials
616 [149,150]. Marine mussels demonstrate robust wet-resistant adhesion under wet
617 conditions. The 3,4-dihydroxy-L-phenylalanine (DOPA) play a key role in this
618 adhesion [151]. The catechol (ortho-dihydroxypheny) group of the DOPA forms
619 strong covalent and noncovalent bonds with various inorganic/organic/metallic
620 surfaces [152,153]. Bioinspired from the mussel adhesion mechanism, the approach
621 which introduced the catechol moiety into chitosan backbone has been developed for
622 its high elasticity and less toxicity [154]. Ryu and collaborators realized an robust
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623 tissue adhesive hydrogels by dissolving separately the catechol-functionalized

624 chitosan (CHI-C) and the thiol-terminated Pluronic (Plu-SH) in PBS solution at a pH
625 of 7.4, and then mixing them for 1 h at 4◦C [155]. At basic pH and body temperature

626 (37◦C), the hydrogels could be crosslinked via the reaction of oxidized catechol with
627 the primary amino groups of the chitosan and with thiol groups of the thiolated
628 Pluronic via a Michael addition reaction or a Schiff-base formation reaction (Fig. 5a).
629 As shown in Fig. 5b, the detachment stress value of CHI-C/Plu-SH hydrogel
630 was15.0±3.5 kPa, which was the highest adhesion forces compared with other
631 polymers. These catechol-functionalized chitosan and Pluronic composite hydrogels
632 are expected to apply in novel tissue adhesives for drug delivery and tissue
633 regeneration for its strong adhesiveness.
634
635
636
637
638
639
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641
642
643 Fig. 5 Adhesive hydrogel a) Injectable hydrogel consisting of catechol-conjugated chitosan/and
644 thiol-terminated Pluronic. Reproduced from ref. 155 with permission from the American Chemical
645 Society, copyright 2011. b) Detachment stress of various polymers. Reproduced from ref. 155 with
646 permission from the American Chemical Society, copyright 2011.
647 3.3 High strength hydrogel

648 A normal structure hydrogel will break up under low stress due to the heterogeneous
649 network structure, so it limit the applications of hydrogel mostly in drug delivery
650 systems, sensors, and smart switching device [156]. To date, more and more studies
651 on hydrogels focused on reinforcing the mechanical strength. Many efforts have been
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652 made to optimize three-dimensional network structures, such as Double network (DN)
653 hydrogels, Nanocomposite (NC) hydrogels, tetra-PEG hydrogels [157], and
654 Macromolecular microsphere composite (MMC) Hydrogels etc. Herein, several

655 strategies on improvement the mechanical properties of the chitosan hydrogel will be
656 introduced.
657 3.3.1 Double network (DN) hydrogel
658 Double network (DN) hydrogel is composed of two kinds of polymer networks with
659 strong asymmetric structure. The basic framework consist of rigid and brittle polymer
660 served as sacrificial bonds effectively dissipates energy, and the second network
661 compose of soft and flexibility polymer maintained hydrogel integrity during
662 deformational process [158,159] (Fig. 6a). Compared to general interpenetrating
663 network hydrogels, DN hydrogels exhibit excellent mechanical properties (Fig. 6b):
664 rigidity (elastic modulus of 0.05–1.0MPa), strength at break (fracture tensile stress of
665 0.2–10MPa; strain of 100–6000%), and toughness (tearing fracture energy of 100–
666 15000 Jm−2) [160-162]. The increasing toughness of DN hydrogel is due to the
667 internal fracturing of the brittle network effectively dissipates energy and the elasticity
668 network allows it to return to its original configuration after deformation [158,159].
669 In the process of construction the hybrid DN hydrogels, the chitosan is not a
670 preferable choice, because the low solubility and high viscosity of long-chain CS
671 generate forming the brittle and weak CS hydrogels. To overcome this problem, Yang
672 group produced the composite PAM-CS hydrogels by integrate short-chain CS into a
673 covalent PAM network (Fig. 6c), and then yielded various hybrid DN hydrogels
674 (PAM-CS DN hydrogels) via adjusting soaking solutions [163]. This DN hydrogels
675 display high tensile strength (≈2 MPa), ultrahigh toughness (fracture energy: ≈10
676 kJ m−2), self-recovery property (exceed 90% recovery efficiency after relaxing for 4
677 h), excellent load-bearing capacity, anti-crash capacity, and fatigue resistance (Fig. 6d,
678 5e). The excellent mechanical properties of PAM-CS DN hydrogels originated from
679 the formation of CS microcrystalline and chain-entanglement networks.
680 Zhang team fabricated the DN hydrogel with thiolated chitosan (Chitosan-NAC)
681 and oxidized dextran (Odex). The chemically cross-linked domains were established
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682 through Schiff base formation and disulfide bonding [164]. In this system, the first
683 crosslinked network was created by the relatively fast formation of imine bond when
684 the free -NH2 groups on the chitosan-NAC reacted with Odex, and the second

685 network composed of disulfide bonds during the slower interaction of the -SH groups
686 along the chitosan-NAC molecular chains. Collectively, the results of burst strength
687 testing and the rheological measurements congruously approved that
688 Odex/Chitosan-NAC hydrogel has mechanically strong with a short tgel and reaction
689 time span.
690
691
692
693
694
695
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697
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699
700
701 Fig. 6 DN-hydrogel a) By combining different network materials, tough double-network materials
702 can be created. Reproduced from ref. 158 with permission from Science, copyright 2014. b) Photo
703 of a tough double-network hydrogel containing 90 wt% of water. Reproduced from ref. 158 with
704 permission from Science, copyright 2014. c) Mechanisms of converting the composite hydrogel
705 into hybrid DN hydrogels. Reproduced from ref. 163 with permission from Wiley-VCH, copyright
706 2016. d) The mechanical properties of PAM-CS-A DN hydrogels. Reproduced from ref. 163 with
707 permission from Wiley-VCH, copyright 2016. e) The mechanical properties of PAM-CS-S DN
708 hydrogels. Reproduced from ref. 163 with permission from Wiley-VCH, copyright 2016.
709 3.3.2 Nanocomposite (NC) hydrogel
710 Nanocomposite (NC) hydrogel compose of polymeric hydrogels and a

711 water-swellable inorganic clay. In fact, NC gels can withstand high levels of
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712 deformation, not only in the form of elongation and compression, but also bending,
713 tearing, twisting and even knotting [165] (Fig. 7b). The principle of NC gels
714 reinforcing mechanical performance is to use nanoscale inorganic particles as

715 multifunctional crosslinking agent for the polymer chain [166] (Fig. 7a). So far, based
716 on small size effect of zero-dimensional, one-dimensional and two-dimensional

717 materials, a series of chitosan nanocomposite hydrogels have been fabricated.
718 Konwar et al. reported a chitosan(CH)/carbon dots(CD) nanocomposite hydrogel
719 films with improved physico-mechanical properties compared to that of chitosan
720 hydrogel film [167]. The tensile strength (TS) of CH-CD (0.5wt% tea CDs)
721 nanocomposite film increased considerably to 18.6 MPa in comparison to CH
722 hydrogel film (5.1 MPa), and the mechanical strength was attributed by the strong
723 electrostatic interaction between the polymer hydrogel matrix and stiff carbon dots.
724 Rong et al. enhanced the strength of chitosan film by addition of nanowhiskers
725 (CNWs) into chitosan to obtain biocomposite film [168]. Tensile tests of this system
726 indicated the increased tensile of film strength up to 24.4 MPa (10 % CNWs
727 concentration) and Young’s modulus to 858.68 MPa (25% CNWs concentration),
728 while decreased elongation at break to 3.2% (25% CNWs concentration). Good
729 interaction and adhesion, the presence of hydrogen bonding between CNWs and
730 chitosan matrix both improved the performance of biocomposite.
731 Yao group fabricated nacre-like bionanocomposite hydrogel films with high
732 strength from the self-assembly of chitosan–montmorillonite(MTM) hybrid building
733 blocks [169] (Fig. 7c,7d). In their works, the chitosan molecules were coated onto
734 exfoliated MTM nanosheets by strong electrostatic and hydrogen-bonding interactions
735 to yield the hybrid building blocks, so it were fabricated the hydrogel films by
736 sequential dipping coating and the LBL technique. Tensile-strength tests showed that
737 the Young’s modulus and ultimate tensile strength of the chitosan–MTM films were
738 respectively 3–5time and 2–3time higher than that of the conventional film because
739 brick-and-mortar microstructures. An atomic modeling was made further investigation
740 and proved the conclusion described above. Furthermore, the bionanocomposite films
741 had light transmittance and fire resistant properties. This green man-made nacre-like
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742 chitosan–MTM film with high performance was a hopeful material for the substitute
743 of conventional petroleumbased plastics.
744 Yang et al. dispersed graphene oxide (GO) in chitosan solution to synthesis of

745 CS/GO nanocomposites [170]. Electrostatic attraction and hydrogen bonding between
746 CS and GO enhanced interfacial adhesion and improved mechanical performance of

747 the nanocomposite. The tensile strength and Young’s modulus were significantly
748 improved by about 122 and 64%, respectively, by addition of only 1 wt % GO.
749
750
751
752
753
754
755
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759
760 Fig. 7 NC-hydrogel a) Network structure and elongated structure models for the NC gel. In the
761 model, only a small number of polymer chains are depicted for simplicity. Reproduced from ref.
762 166 with permission from Wiley-VCH, copyright 2002. b) NC gels exhibit extraordinary
763 mechanical toughness. Reproduced from ref. 165 with permission from Elsevier, copyright 2007.
764 c) Fabrication of the artificial nacre-like chitosan–MTM bionanocomposite film. Reproduced from
765 ref. 169 with permission from Wiley-VCH, copyright 2010. d) Atomic modeling of the chitosan
766 molecules adsorbing on the MTM surface. Reproduced from ref. 169 with permission from
767 Wiley-VCH, copyright 2010.
768 3.3.3 Macromolecular Microsphere Composite (MMC) Hydrogel

769 The hydrogel make of macromolecular microspheres (MMSs) on the micro- or
770 nanoscale is called microgels or nanogels. These microgels caused the widely
771 attention for their environmentally sensitive and the applications in drug delivery
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772 [171]. However, they are difficult to form bulk hydrogels (macrogels) [172], and the
773 macrogel won't have high mechanical strength even if formed. Gong team
774 successfully increased the mechanical strength of microgels by introduction of

775 macromolecular microsphere composite (MMC) into double-network (DN) hydrogels
776 [173,174]. In their works, the significant swelling properties in acidic medium of
777 dried chitosan microspheres (CSM) to weave prepolymerization solution were utilized
778 constructing a microsphere-structured hydrogel with excellent mechanical properties
779 [175]. Inspired by this consideration, Zhang and colleagues constructed transparent
780 chitosan/polyacrylamide (PAAm) hydrogels (coded as M-Gel) and dark green
781 chitosan/polyacrylamide (PAAm)/polyaniline (PANI) hydrogels (coded as MC-Gel)
782 (Fig. 8). Tension tests exhibited tensile strength of the M-Gel was 30-fold higher than
783 that of pure chitosan gel, and the M-Gel also showed extremely tensile resistance and
784 compression resistance properties. The researchers confirmed that the chitosan
785 microspheres as the unique microscale joint regions reinforced the strength of PAAm
786 hydrogels. Furthermore, MC-Gel not only created the high mechanical strength,
787 ultra-stretchability (strain 600%) and remarkable mechanical stability, but also
788 exhibited conductivity and force sensitivity. These properties gave the MC-Gel
789 potential applications in wearable health monitoring, electronic skin, and nerve
790 regeneration [176,177].
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802 Fig. 8 MMC-hydrogel a) The schematic preparation process of the MC-Gel. Reproduced from ref.
803 175 with permission from Wiley-VCH, copyright 2016. b) M-Gel and MC-Gel pressed by a sharp
804 object. Reproduced from ref. 175 with permission from Wiley-VCH, copyright 2016. c) The

805 pictures of mechanical test of MC-Gel. Reproduced from ref. 175 with permission from
806 Wiley-VCH, copyright 2016
807 4. Summary and Outlook

808 Design and preparation of intelligent hydrogels has become more and more popular
809 field in the material research nowadays. Chitosan based intelligent hydrogels attracted
810 many researcher attention for their biocompatibility and functionality. It can be used
811 as drug delivery matrix and a substitute for multiple-tissue engineering materials
812 [178]. Although successful and fruitful results have been obtained in these fields,
813 however, currently a few chitosan-based gels are applied in practice. There are still
814 many challenges in the following aspects: Firstly, the limitation of characteristic
815 properties with the different interactions fabricated hydrogel result drawback in the
816 applications of the chitosan based gels. The hydrogel fabricated with physical
817 interactions is difficult to precisely control chemical functionalization and degradation
818 or dissolution, leading to inconsistent performance in vivo. In addition, the weak
819 mechanical strength, uncontrolled dissolution property and fast release of drugs
820 always affect the applications range. Compared with physical hydrogels, the presence
821 of free unreacted crosslinker and other auxiliary molecules in the chemical hydrogels
822 could not be completely excluded and may induce toxic effects even if hydrogels are
823 purified before administration [33]. Investigated non-toxic covalent crosslinkers with
824 biocompatibility as alternatives for the chitosan hydrogel and developed the gel
825 combined the characteristics of the physical and chemical hydrogels are important for
826 future studies.
827 Secondly, the main disadvantage of the external stimuli-sensitive hydrogel is that
828 their low responsive rate or slow response time. Rapid responsive capacity of chitosan
829 based hydrogel can be easily achieved with designing thinner and smaller hydrogel
830 which decrease the hydrogel systems mechanical strength [179]. Therefore, it is
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831 necessary to develop the fast-acting hydrogel with excellent mechanical strength.
832 Finally, how to fabricate the multi-functional and highly intelligent chitosan
833 hydrogel is the most challenge to the all of researchers. Construction the biomaterials

834 with stimuli responsive, self-adaptive, and capable of self-healing by biological
835 process liked as organisms is the ultimate goal [180]. In the chitosan gels, hydrogel
836 prepared with a single or several materials can never satisfy this desire, thus
837 composited variety of specific function materials to the system and following the
838 biomimetic principles to prepare hydrogel will lead to success in exploring the new
839 bioinspired chitosan based hydrogels in the future.
840 Acknowledgements
841 This study was funded by the National Natural Science Foundation of China (Grant
842 number: 51522510, 51675513, 51705138 and 51735013), and by Hubei Province
843 Department of Education Scientific Research Project (B2017053).
844
845 Conflict of Interest: The authors declare that they have no conflict of interest.
846
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Graphic abstract

This review puts an emphasis on chitosan intelligent hydrogels. The fabrication and mechanism of
are introduced in this review and the interactions of the formation hydrogels with both physical
and chemical bonds are introduced. Meanwhile, the relationship between the structural characters
and the corresponding functions of stimuli responsive characteristics, self-healing functions and
high mechanical strength properties of the chitosan hydrogels are detailed discussed in the review.

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1 The chitosan hydrogels: From structure to function

3 Jing Fu 1,2, Fuchao Yang 1, Zhiguang Guo1,3*

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

36 undersized molecules and release of entrapped molecules in a controlled manner [2-4].

New Journal of Chemistry Accepted Manuscript

76 Elsevier, copyright 2004.

87 delivery of biological therapeutics with thermo-setting chitosan hydrogel was reported

New Journal of Chemistry Accepted Manuscript

91 were contributed to gel formation. Wei et al. fabricated a novel chitosan-based

116 2. Formation of the chitosan hydrogels

New Journal of Chemistry Accepted Manuscript

130 2.1 Physical hydrogels

New Journal of Chemistry Accepted Manuscript

149 (such as ZnO, Ag etc.) into chitosan hydrogels to generate organic-inorganic

175 structure, hydrophobicity/hydrophilicity balance, stereo-regularity, flexibility, and

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

251 cells during the injection process.

252 2.2 Chemical hydrogels

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

Gutaraldehyde Chitosan [82]

Salicyladehyde Chitosan [83]

Formaldehyde Chitosan [84]

Oxidized sodium N-carboxyethyl

New Journal of Chemistry Accepted Manuscript

Oxidized pectin Chitosan [90]

telechelic Chitosan [94]

New Journal of Chemistry Accepted Manuscript

Diepoxy-PEG Chitosan [103]

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

388 3. Chitosan intelligent hydrogels

395 3.1 Stimuli-responsive hydrogels

404 swelling behaviors, network structure, permeability, or mechanical strength) of

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

464 (poly(N-isopropylacrylamide)) or PNVCL [118] (poly(N-vinylcaprolactam)) are

New Journal of Chemistry Accepted Manuscript

468 in the hydrogels can adjust the thermosensitive capacity.

New Journal of Chemistry Accepted Manuscript

524 Chitosan/polycation hydrogel and amphoteric chitosan/polyanion hydrogel were

New Journal of Chemistry Accepted Manuscript

528 chitosan/polyallylamine hydrogel [134], chitosan/poly (vinyl alcohol) hydrogel [135]

New Journal of Chemistry Accepted Manuscript

557 representation of thermosensitive networks of physical gelation driven by hydrophobic

563 3.2 Bio-inspired hydrogels

574 (disulfide), boron–oxygen bonds (phenylboronate ester), carbon–nitrogen bonds

New Journal of Chemistry Accepted Manuscript

604 capability to the CEC/OSA/ADH hydrogel under physiological conditions. This