Carbohydrate Polymers 199 (2018) 445–460

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Carbohydrate Polymers

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Chitosan based hydrogels and their applications for drug delivery in wound dressings: A review T ⁎ Hamid Hamedi, Sara Moradi, Samuel M. Hudson, Alan E. Tonelli

Textile Engineering Chemistry and Science, Fiber & Polymer Science Program, College of Textiles, North Carolina State University, Raleigh, North Carolina 27606-8301, United States

ARTICLE INFO ABSTRACT

Keywords: Advanced development of chitosan hydrogels has led to new drug delivery systems that can release their active Chitosan hydrogel ingredients in response to environmental stimuli. This review considers more recent investigation of chitosan Wound dressing hydrogel preparations and the application of these preparations for drug delivery in wound dressings. Drug delivery Applications and structural characteristics of different types of active ingredients, such as growth factors, na- Growth factor noparticles, nanostructures, and drug loaded chitosan hydrogels are summarized. Nanoparticles

1. Introduction 2012), pullulan (Li et al., 2011; Wong et al., 2011) and/or synthetic polymers like polyvinyl alcohol (Kokabi et al., 2007; Razzak, 2001; Hydrogels are three-dimensional, cross-linked networks which can Yang et al., 2008), polyacrylamide (Ezra et al., 2009; Risbud & Bhonde, absorb and retain significant amounts of water, without dissolving or 2000; Rosiak et al., 1983) and polyethylene glycol (Ajji et al., 2005; losing their three dimensional structures (Ahmed, 2015; Kashyap, 2005; Gupta et al., 2011; Lih et al., 2012) form hydrogels. Y.B. et al., 2008). The gelation and biodegradation are two key factors Hydrogels are classified into two categories: chemical or permanent affecting the fate of cells (Li et al., 2012). Moreover, some hydrogels gels that have covalently bonded cross-linked networks (replacing hy- have antibacterial and antifungal activities (Jaya kumar et al., 2011), drogen bonds by strong and stable covalent bonds) and physical or and these properties could be useful for wound dressings and accel- reversible gels whose networks are held together by molecular en- erating the wound healing process (Ueno et al., 2001). Polymers that tanglements, and/or secondary forces including ionic, hydrogen form hydrogels may have hydrophilic or hydrophobic functional bonding or hydrophobic interactions (Ahmed, 2015). In physically groups. Hydrophilic functional groups, such as hydroxyl (−OH), amine cross-linked gels, dissolution is prevented by physical interactions,

(NH2) and amide (−CONH−CONH2) enable the hydrogel to absorb which exist between different polymer chains (Hennink & Nostrum, water leading to hydrogel expansion, which is known as swelling. 2002). Hydrogels can be formulated in a variety of physical shapes, During swelling, the cross-linked structure of hydrogels prevents com- including slabs, microparticles, nanoparticles, coatings, and films plete destruction of the hydrogel cross-links and dissolution. Hydrogels (Hoare & Kohane, 2008). with hydrophobic chains such as poly lactic acid have lower water The most important property of hydrogels is their biocompatibility, swelling capacity than hydrophilic lattices. Hydrogels can be prepared which can be defined as the ability of a material to be in contact with from synthetic or natural polymers, involving a wide range of chemical bodily organs with minimum damage to the surrounding tissues and compositions and with different mechanical, physical and chemical without triggering undesirable immune responses (Caló & properties. Natural polymers such as alginate (Balakrishnan et al., Khutoryanskiy, 2015). In some cases this property has been taken ad- 2005; Kamoun et al., 2015; Kim et al., 2008; Murakami et al., 2010), vantage of in wound dressings, because after healing it prevents ex- chitosan (Milosavljević et al., 2010; Racine et al., 2017), hyaluronic cessive tissue granulation and scar formation (Chung et al., 1994), acid (Luo et al., 2000; Segura et al., 2005; Silva et al., 2017) cellulose though they can be hard to handle, may be difficult to load with drugs (Abd El-Mohdy, 2013; Chang et al., 2009; Maneerung et al., 2008; and sterilize, and usually have low mechanical strength (Huffman, Sannino et al., 2009), starch (Pal et al., 2006; Pal et al., 2006; Zhai 2012). In spite of these disadvantages, hydrogels have many applica- et al., 2002), ulvan (Alvesab et al., 2012; Morelli & Chiellini, 2010), tions in different fields including tissue engineering, pharmaceuticals gelatin (Draye, Delaey, Voorde, Bulcke, Reu et al., 1998; Wang et al., and biomedical engineering, in the forms of wound dressings (Benamer

⁎ Corresponding author. E-mail address: [email protected] (A.E. Tonelli). https://doi.org/10.1016/j.carbpol.2018.06.114 Received 19 March 2018; Received in revised form 25 June 2018; Accepted 26 June 2018 Available online 28 June 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved. H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460 et al., 2006; Kokabi et al., 2007; Lu et al., 2010), drug delivery vehicles degree of deacetylation (97.5%) lead to higher positive charge density, (Hoare & Kohane, 2008; Qiu & Park, 2001; Xinyin, 2003), implants which confers stronger antibacterial activity than moderate degree of (Refojo & Leong, 1981; Stasica et al., 2000; ZZZZZ, 2018), and in- deacetylation (83.7%) against Staphylococcus aureus at pH 5.5 (Kong jectable polymeric systems (J.P. & T.H., 2006; Macaya & Spector, 2001; et al., 2008b). It was reported that polydispersity is a useful quantity Shibata et al., 2010; Yu & Ding, 2008). which can clarify the degradation mechanism (Chen et al., 1997).The Chitosan is one of the most widely used materials for making hy- decrease of polydispersity as degree of deacetylation increases could drogels, and has been considered for wound dressing applications. It appear as surprising since the deacetylation reaction does not degrade has excellent biocompatibility, low toxicity and immune-stimulatory chitosan chains (Schatz et al., 2003). Min et al. (Tsai et al., 2008) have activities (Li, Rodrigues et al., 2012; Souza et al., 2009). Due to these studied factors that are effective on chitosan polydispersity, such as, properties, chitosan shows good biocompatibility and positive effects solution temperature and solution concentration of chitosan. They also on wound healing. It can also accelerate repair of different tissues and explained why the polydispersities of chitosan nanoparticles obtained facilitate contraction of wounds (Jaya kumar et al., 2011). The Cyto- by ultrasonic treatment or by mechanical shearing were similar. The compatibility of chitosan has also been studied and shown that it does structures of chitosan hydrogels can affect their swelling behaviors, not have acute cytotoxic effect (Gonçalves Ferreira et al., 2016). On the because swelling behavior reflects the amount of network porosity and other hand, as a natural based compound, chitosan may be con- mesh size of the network, which may also indicate its drug release taminated by organic and inorganic impurities, and presents broad behavior (Berger et al., 2004). Porosity of chitosan membranes could be polydispersity. Chitosan is poorly soluble, except in acidic medium, modified by crosslinking with some materials such as glutaraldehyde which makes analyses difficult to perform (Croisier & Jérôme, 2013). (Krajewska, 2001). Chitosan also has been used in commercial wound dressings such as Chitosan under acidic condition also has a high capacity for en- ™ ChitoSam (sam medical, 2018), ChitoGauze XR pro (North American trapping various metal ions including (Ni2+,Zn2+,Co2+,Fe2+,Mg2+ Rescue, 2018), ChitoFlex (H.M.T. Inc., 2007), and Axiostat® (AXIO, and Cu2+)(Kurita, 1998). Rainaudo et al. (Mazeau et al., 2000) have 2018) which are high-performance hemostatic dressings. studied chitosan chain stiffness, and concluded that chitin and chitosan This review emphasizes on chitosan hydrogels and their applica- are semi-rigid polymers characterized by a persistence length (asymp- tions in wound dressing and drug delivery. Initially chitosan and its totic value obtained at high degree of polymerization) that depends properties are described and different kinds of hydrogel preparation moderately on the degree of acetylation of the molecule. methods are discussed. In the second section the utilization of chitosan hydrogels are summarized. Finally, utilization of some drugs such as 2.1. Sources of chitosan growth factors, nano particles and chemicals in chitosan hydrogels are described. Chitin, poly (β-(1→4)-N-acetyl-D-glucosamine) is a natural poly- saccharide, first identified in 1811 by French chemist Henri Braconnot 2. Chitosan (Arias et al., 2004). Chitin forms a part of the extracellular matrix of certain living organisms as a proteoglycan. It is the most abundant Due to its reactive amino groups, chitosan is the only natural ca- polymer after cellulose. This biopolymer is synthesized by an enormous tionic polymer and has many commercial applications: it accelerates number of living organisms among which are insects (cuticles) and wound healing, is antimicrobial, anticoagulant, antibacterial, anti- crustaceans (skeletons), e.g., crab, shrimp and lobster. Cephalopod by- fungal, anti-tumor, and haemostatic [62,63]. The generic term chitosan products are a valuable source of chitin, polyunsaturated acids, and describes a range of presumably pure poly-(beta-1-4) N-acetyl-D-glu- collagen. (Koueta, 2014). Chitin is also extracted from the exoskeleton cosamine materials (shown in Fig. 1) whose properties are highly de- of cephalopod species, such as squid, cuttlefish and octopi (Arrouze pendent on its degree of deacetylation; average molecular weight, et al., 2017; Jothi & Nachiyar, 2013; Kurita et al., 1993; Shanmugam polydispersity, and its structure. et al., 2016). (See Fig. 1). (Zhou et al. (2008)) investigated the effect of molecular weight and Chitosan, which was discovered and named in 1859 by Roget degree of deacetylation on chitosan hydrogels and concluded that these (Patrulea et al., 2015), is obtained by partial deacetylation of chitin two parameters affected the pH values, turbidity, viscosity, and ther- under alkaline conditions and is the most important chitin derivative in mosensitive characteristics of the product hydrogels. Degree of deace- terms of applications (Rinaudo, 2006b). (See Fig. 1). The physico- tylation affects chitosan antimicrobial activity (Chung et al., 2004; Liu chemical characteristics of the obtained chitosan are closely related to et al., 2001); on the other hand chitosan microspheres with a high the taxonomy of the marine sources (Rhazi et al., 2000). Chitosan can

Fig. 1. Chitosan structure.

446 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460 also be produced by enzymatic rather than alkali treatment at high chitosans (testing of chitosans in vivo and in vitro are often performed at temperatures (Cai et al., 2006; No & Meyers, 1995). Chitosan can be neutral pH-values). enzymatically degraded by chitinases and chitosanases but these en- Sorlier et al. (Sorlier, Viton, and Domard, (2002)) demonstrated that zymes are unavailable in bulk quantities for commercial applications. It when the degree of dissociation (α) in solution increases, the role of the has been reported that β-glucosidase from almond emulsin can hydro- cationicity of the amine groups, which depends on the degree of acet- lyze chitin substrates owing to an existing chitinase in the enzyme ylation, plays a more important role on the behavior of the polymer preparation (Zhang & Neau, 2011). It has been reported that chitinases chains. They showed that molecular weight and degree of deacetylation are not responsible for the in vitro degradation of chitosans in human have important effect on chitosan solubility. serum (Vårum et al., 1997). Enzymatic degradation minimizes adverse Solubility of chitosan will be enhanced by decreasing its molecular chemical modifications of products and promotes their biological ac- weight. Molecular weight changes the content of N-acetylglucosamines tivities. However, higher costs of hydrolytic enzymes limit the appli- units in chitosan, which will have intramolecular as well as an inter- cation of enzymatic methods. To reduce this production cost, reuse of molecular influences, resulting in different chitosan conformations. hydrolytic enzymes may be recommended (Kim & Rajapakse, 2005). However, improving solubility by controlling deacetylation comes at Chitosan preparation from fungal cell walls with fermentation the cost of low yield (Kurita et al., 1991). Sogias et al.. (Sogias, Khu- technology has become an alternative economical way for the pro- toryanskiy, and Williams, (2010)) showed that break down of chitosan duction of this polymer. It can be found in the cell wall of certain crystallinity will expand the range of chitosan solubility. They studied groups of fungi, particularly zygomycetes (Nwe et al., 2002; Tayel et al., two approaches for improving chitosan solubility, physical and che- 2010). There are several advantages of using these fungi to produce mical methods. In their chemical approach, re-acetylation improved chitosan. The most important is that the cell wall of zygomycetous fungi chitosan solubility up to pH = 7.4. The physical approach included the contains a large quantity of chitosan and the physicochemical proper- use of additives, such as urea, and guanidine hydrochloride, with the ties of this chitosan can be manipulated and standardized by controlling abilities to disrupt intra- and inter macromolecular hydrogen bonding. − − the parameters of fermentation. For example, different molecular In the presence of 8 mol L 1 urea and 1.5 mol L 1 guanidine hydro- weight chitosans can be produced when these fungi are grown on at chloride the solubility of chitosan shifted to pH = 8.0 and 6.5, respec- different pHs and on media with different compositions (Tan et al., tively. 1996). Sufficient amounts of chitosan have been identified in Mucor Introducing small chemical groups to the chitosan structure, such as rouxii (White et al., 1979), phycomyces, and saccharomyces (Arcidiacono alkyl (hydroxypropyl chitosan (Xie et al., 2002)) or carboxymethyl & Kaplan, 1992). In another study, chitosans with high degree of dea- groups can drastically increase the solubility of chitosan (Jayakumar cetylation (above 97%) were extracted from R.miehei and M.racemosus et al., 2010; Pillai et al., 2009). A simple and improved method of (Tajdini et al., 2010). preparing highly soluble chitosan (half N-acetylated chitosan) was de- An investigation was performed to evaluate the chitosan char- veloped using a series of chitosan samples of low molecular weights acterization produced by several species of fungi. Comparison of fungal (Kubota et al., 2000). chitosan with a commercial one derived from crab shells showed lower degree of deacetylation, viscosity and molecular weight of the fungal 2.3. Toxicity chitosan. Chitosan with a low molecular weight reduces the tensile strength and elongation of the chitosan membrane, but increase its There are various studies on chitosan toxicity. Scientists have done permeability. Fungal chitosan may have potential medical and agri- some studies on chitosan toxicity in guinea pigs (Aspden et al., 1997), cultural applications (Pochanavanich & Suntornsuk, 2002). This is in frog, human nasal palate tissue (Aspden et al., 1994; Aspden et al., general agreement with the findings of Yen et al. (Yen & Mau, 2007; 1995), and the nasal membranes of rats (Aspden et al., 1996). In all of Yen, Yang, and Mau, (2009)). the tests, toxicity was negligible. Ribeiro et al. (Ribeiro et al. (2009)) studied the in vitro cytotoxicity of chitosan hydrogels tested with dermal 2.2. Solubility of chitosan fibroblasts obtained from rat skin. Their cell viability study showed that the hydrogel and its degradation by-products are non-cytotoxic. His- Due to the high cohesive energy of chitin, which is related to strong tological analysis also revealed lack of a reactive or a granulomatous intermolecular interactions from hydrogen bonds, chitin is insoluble in inflammatory reaction in skin lesions with chitosan hydrogels, and the many typical solvents, such as water, dilute acids and alcohols (Zargar absence of pathological abnormalities in the organs supported the local et al., 2015). This is a major problem that limits the development of and systemic histocompatibility of this biomaterial. processing and usage of chitin. in vivo chronic toxicity studies reported by Carreño-Gómez et al. Chitosan, the most important derivative of chitin, is soluble in dilute (Carreño-Gómez & Duncan, 1997) have shown that intravenous ad- aqueous acidic media at a degree of deacetylation of 50% and higher ministration of chitosan (4.5 mg/kg/day for 11 days) to rabbits did not (depending on the origin of the polymer) due to its primary amino lead to any abnormal changes. Data on chitosan toxicity from human groups that have a pKa value of 6.3. Solubilization occurs by protona- studies in general are quite limited. Gades et al. (Gades & Stern, 2003) tion of the –NH2 group of the D-glucosamine repeating unit, whereby reported that quantities exceeding 4.5 g chitosan taken daily by human the polysaccharide is converted to a polyelectrolyte in acidic media. volunteers did not result in toxic effects. Even higher oral levels of up to Solubility of chitosan is usually examined in acetic acid by dissolving it 6.75 g were reported as safe. It has been reported that short-term in 1% or 0.1 M acetic acid (Rinaudo, 2006a). Rinaudo et al.. (Rinaudo, human trials of up to 12 weeks have shown no clinically significant Pavlov, and Desbrie`res, (1990)) demonstrated that the amount of acid symptoms, including no evidence of an allergic response (Tapola et al., needed depends on the quantity of chitosan to be dissolved. The con- 2008). Chitosan mouthwash safety was evaluated through Ames, MTT centration of protons needed is at least equal to the concentration and V79 chromosomal aberration assays. The chitosan mouthwash of − NH2 units involved. As the degree of protonation is progressively possessed lower toxicity and higher antimicrobial activity than the increased, so is the solubilization of chitosan. The effect of depoly- commercial mouthwash tested. Furthermore, while it is capable of merization on the solubility of the chitosans with different degree of completely inhibiting the two selected pathogenicity markers (strep- deacetylation at neutral pH-values was investigated by Vårum et al tococci and enterococci), contrary to the commercial mouthwash, it did (Varum et al., 1994). They emphasized that the solubility differences not cause major reductions in the viability of the normal oral microflora between chitosans with different degree of deacetylation may have (Costa et al., 2014). profound influence on accessibility of chitosans to enzymes (many en- Ravindranathan et al. (Ravindranathan, Koppolu, Smith, and zyme assays are performed at pH 6.5) and the biological effects of Zaharoff, (2016)) Studied purified, low endotoxin chitosan, and

447 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460 determined that viscosity/molecular weight and degree of deacetyla- pepsin, an enzyme that breaks down proteins into smaller peptides, tion within the ranges of 20–600 CP and 80–97%, respectively, have no enhanced the ability of the glycan to activate the inflammasome (Bueter impact on chitosan’s immunoreactivity. Endotoxin contamination was et al., 2011). expected to be a major factor influencing immunoreactivity. Their re- Biodegradability of chitosan has been studied by many researchers. sults indicated that only endotoxin content and not degree of deace- It was found that lysozyme (Onishi & Machida, 1999), proteases (Rao & tylation or viscosity influenced chitosan-induced immune responses. Sharma, 1997) and porcine pancreatic enzymes (Verheul et al., 2009) Their data also indicated that low endotoxin chitosan (< 0.01 EU/mg) are able to degrade Chitosan under specific conditions. Pectinase iso- with viscosities of 20–600 cP and 80%–97% degree of deacetylation are zyme from Aspergilus niger has also been shown to digest chitosan at essentially inert. This study highlights the need for more complete low pH providing lower Mw chitosans (Kittur et al., 2003; Kittur et al., characterization and purification of chitosan in preclinical studies be- 2005). Connell et al. (McConnell et al., 2008) used human fecal pre- fore being used in clinical application. parations and showed significant degradation of chitosan films, glu- Baldrick (Baldrick (2010)) implies that chitosan can be used as a taraldehyde crosslinked films, and tripolyphosphate crosslinked films. safe pharmaceutical excipient for non-parenteral and non-blood con- Brenner et al. (Brenner, Hostetter, and Humes, (1978)) demonstrated tact. Based on the available data, safe use of chitosan as a parenteral that many enzymes seem to show an activity against chitosan, at least in excipient is not clear. Its use as a medical device to control bleeding vitro, but derivatives could give indigestible molecules. To be clinically relies on the haemostatic biological nature of the material and studies viable, these compound particles should remain small enough (< 42 Å have demonstrated local findings, such as blood coagulation, thrombus radius for neutral molecules) to be renally excreted. The bio-distribu- formation and platelet adhesion/aggregation. In spite of some in vitro tion is controllable through the route of administration, dosage form studies related to cytotoxicity findings (Baldrick (2010)), there are re- and chitosan characteristics, i.e., a film/dense matrix will persist at the ports of non-toxic usages of chitosan in pharmaceuticals, such as to stop site of administration due to the physical characteristics. The de- bleeding (Dowling et al., 2011; Horio et al., 2010; Valentine et al., gradation of the matrix is controllable through crosslinking modifica- 2010). tion of the amine groups. Intravenous distribution can be adjusted by particle size (< 100 nm), and molecular weight can determine the 2.4. Biomedical properties stability of the particle (Kean & Thanou, 2010).

The degradation rate of chitosan can be controlled by changing its degree of deacetylation and/or its molecular weight (Tomihata & Ikada, 2.5. pH sensitivity 1997). Many researches have been done to prove the degraded products of chitosan are nontoxic, non-immunogenic, and non-carcinogenic The numerous amine groups on the chitosan backbone confer a pH- (MuzzareUi, 1993). Risbud et al. (Risbud & Bhonde, 2000) reported dependent solubility, which has been utilized to fabricate biosensors characterization, biocompatibility, and release properties of poly- and drug delivery systems. Chitosan properties can be conveniently acrylamide/chitosan hydrogels and showed that chitosan possess ex- altered by derivatization of its hydroxyl and amino groups (Zhang et al., cellent biocompatibility and bioabsorbability. In this research, in vitro 2012). The amino groups of chitosan are ionized in acidic buffers, cytotoxicity studies using a colorimetric assay for assessing cell meta- which contribute to the electrostatic repulsion between adjacent io- bolic activity, membrane permeability, and lysosomal activity of cells nized –NH2 groups and lead to chain expansion and eventually increase (MTT and NR assays) showed no toxic effects on different cells in up to the water absorption of the chitosan gels. The swelling degree of the 40% hydrogel extract. ionic pH-sensitive hydrogel is mainly determined by the degree of io- The antimicrobial properties of chitosan and its derivatives have nization (Guanghua et al., 2008). Some attempts have been made for attracted great attention from researchers (Kong et al., 2010). Anti- regulating chitosan pH-sensitivity by adding different additives or microbial activity of chitosan has been demonstrated against many grafting or mixing with other polymers and preparing Polyelectrolyte bacteria, filamentous fungi, and yeasts. Chitosan has a wide spectrum of complexes with anionic polymers (Krishna Rao et al., 2006; Meng et al., activity and high killing rates against gram-positive and gram-negative 2010; Qu et al., 1999; Qu et al., 2000). The nature of the pH response of bacteria (Ueno et al., 1997), with low toxicity toward mammalian cells. chitosan and its derivatives has been described by several researchers. Antibacterial effects of chitosan are reported to be dependent on its Islam et al. (Islam & Yasin, 2012) synthesized a pH sensitive blend molecular weight (Jeon et al., 2001). To prove this idea, No et al. (No, based on chitosan and poly vinyl alcohol (PVA) using a nontoxic cross Park, Lee, and Meyers, (2002)) examined the antibacterial activity of linker for a drug delivery system. They concluded that the pH sensi- six chitosans and six chitosan oligomers with widely different molecular tivity and biocompatibility properties of this chitosan/PVA hydrogel weights against gram-negative and gram-positive bacteria. They made it suitable for medicinal and pharmaceutical applications. Anti- showed that chitosan markedly inhibited the growth of most bacteria microbial activity of chitosan is also pH dependent, it has been reported tested, although their inhibitory effects differed with molecular weight that chitosan displayed antibacterial activity only in an acid environ- and the particular bacterium. Chitosan generally showed stronger ment (Helander et al., 2001). bactericidal effects for gram-positive bacteria than for gram-negative bacteria. The reason for this difference is unclear, but Y. J. Jeon et. al (Jeon et al., 2001) showed the oligosaccharides as well as chitosan 3. Chitosan hydrogel preparation methods exhibited better inhibitory effects against Gram-positive compared with Gram-negative bacteria. Thus, it might be reasonable that chitosan 3.1. Chemical Cross linking (permanent hydrogels) showed remarkable inhibitory effect against the growth of lactic acid bacteria which are Gram-positive. Chemically cross-linked hydrogels are formed by covalent linking of Sizes of chitosan particles play an important role in inflammasome the chitosan macromers, where the bond formation is irreversible. The activation. The inflammasome is a cytosolic complex which is re- cross-linking of natural and synthetic polymers can be achieved through sponsible for the activation of inflammatory responses, such as IL-1β. the reaction of their functional groups (such as OH, COOH, and NH2) Glycans passed through filters of defined size, showed that the smallest with cross-linkers (Liu et al., 2014; Milosavljević et al., 2010; Singh size fraction (< 20 μm) induced the most cleavage of pro-IL-1β and et al., 2006; Zhang et al., 2005). There are a number of methods re- release of the mature cytokine. The larger-sized fractions also had some ported in the literature to obtain chemically cross-linked permanent bioactivity, which may have been due to some smaller particles that hydrogels. The following section reviews the major chemical methods failed to pass through the filters. Partial digestion of chitosan with used to produce chitosan hydrogels.

448 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Fig. 2. Chemical Cross Linker Structures.

3.1.1. Chemical cross-linkers biodegradability and pharmaceutical effects of the genipin-crosslinked Cross-linkers are molecules with at least two reactive functional hydrogels were investigated, and it was concluded that genipin may be groups that allow the formation of bonds between polymeric chains. To well suited for clinical usage (Kitano, 2006; Muzzarelli, 2009a; Yaoa date, the most common cross-linkers used to obtain chitosan hydrogels et al., 2005; Zhang et al., 2006). Effects of genipin cross-linking of are di-aldehydes, such as glutaraldehyde (Milosavljević et al., 2010; chitosan hydrogels on cellular adhesion and viability were investigated. Zhang et al., 2005), formaldehyde (Singh et al., 2006), or ethylene It was found that the cross-linked hydrogels did not induce cytotoxic glycol di-glycidyl ether (EGDE) (Liu et al., 2014), genipin (Mi et al., effects and significantly improved the cell adhesion and viability on 2000; Muzzarelli, 2009b) and others (Draye, Delaey, Voorde, Bulcke, hydrogel surfaces (Gao et al., 2014). Genipin has also been shown to act Bogdanov et al., 1998; Hennink & Nostrum, 2002; Huffman, 2012) (See significantly as an anti-inflammatory and anti-angiogenesis agent, and Fig. 2). protected the hippocampal neurons from the toxicity of Alzheimer’s Among these mentioned cross-linkers, reaction of aldehydes with amyloid beta protein (Kooa, 2004). chitosan is well-documented; the aldehyde groups form covalent imine bonds with the amino groups of chitosan, due to the resonance estab- 3.1.2. Photo crosslinking lished with adjacent double ethylenic bonds (Monteiro & Airoldi, Photo-crosslinking, a type of chemical crosslinking, is performed in 1999). Di-aldehydes allow direct reaction in aqueous media, under mild the presence of ultraviolet (UV) light and a chemical photo initiator conditions and without the addition of auxiliary molecules, such as (PI). A variety of natural and synthetic macromers have been modified reducers, which is advantageous with respect to biocompatibility. to formed hydrogels by photo activation of the coexisting photo in- However, the main drawback of di-aldehydes, such as glutaraldehyde, itiators, such as methacrylated or aryl azide modified macromers is that they are generally considered to be toxic. It is reported that (Rickett et al., 2011). The degree of crosslinking reaction depends on glutaraldehyde is cytotoxic even at low doses and glutaraldehyde UV irradiation time and increases with the increase of exposure time. polymerization may release glutaraldehyde residues during storage or This lead to a hydrogel with higher mechanical properties and lower sterilization (Chang et al., 2004; MacDonald & Pepper, 1994). Glutar- swelling ratio (Ik et al., 2016). The polymerization reaction can also aldehyde is known to be neurotoxic, its fate in the human body is not controlled by adjusting the distance from the UV light source (Ahmadi, fi fully understood. Therefore, even if hydrogels are puri ed before ad- Oveisi, Mohammadi Samani, & Amoozgar, 2015). Obara et al.. (Obara ministration, the presence of free unreacted di-aldehydes in hydrogels et al. (2003)) prepared a photo cross-linkable chitosan aqueous solution ff could not be completely excluded and may induce toxic e ects. This including fibroblast growth factor-2 (FGF-2) by using ultraviolet light could impair the biocompatibility of the cross-linked products (Berger, (UV) irradiation to result in an insoluble, flexible hydrogel. A viscous Reist, Mayer, Felt, Peppas et al., 2004; Chen et al., 2004). azide-chitosan-lactose aqueous solution was converted into an insoluble In order to obtain non-soluble hydrogels for controlling drug release hydrogel within 10 s upon UV-irradiation at a lamp distance of 2 cm and pharmaceutical applications, researchers have been attempting to through crosslinking of the azide and amino groups of the azide-chit- fi nd a cross-linking agent with low cytotoxicity (Chen et al., 2004; Cui osan-lactose molecules. The results showed that the growth factor FGF- et al., 2014; Delmar & Bianco-Peled, 2015). Genipin is a natural cross- 2 molecules remained biologically active, and were released from the linking agent extracted from geniposide, an iridoid glucoside isolated chitosan hydrogel upon in vivo biodegradation. Application of the from the Genipa fruits (Tsai et al., 1994). Genipin has been reported to chitosan hydrogel significantly induced wound contraction and ac- bind with biological tissues (Sung et al., 2001) and biopolymers, such as celerated wound closure. chitosan and gelatin (Bigi et al., 2003; Mi et al., 2005), leading to Among the photo initiators, Irgacure2959 caused minimum cyto- ff covalent coupling. It works as an e ective cross-linker for polymers toxicity (cell death) over a broad range of mammalian cell types and containing amino groups (mostly used for chitosan crosslinking) (Sung species (Hu & Gao, 2008). Pluronic-chitosan hydrogels used as a dres- et al., 1999). It has been found that the reaction rate of genipin with sing for ulcers wounds were fabricated, and the release of growth factor fi amino group containing biomaterials is signi cantly slower than that of was investigated. Photo-irradiation was applied to chemically cross-link glutaraldehyde (Moura et al., 2007; Sung et al., 2000). The reaction the hydrogel. Before irradiation by UV, a photo-initiator, Irgacure2959 between chitosan and genipin (See Fig. 3) is well understood for a (0.1%, w/w), was added to the hydrogel mixture to form physical hy- variety of experimental conditions and has no cytotoxicity for human drogels. Release profiles of rhEGF growth factor from hydrogel net- and animal cells (Muzzarelli et al., 2015). Biocompatibility, works were evaluated with modification of the chitosan blend ratios and photo-irradiation time. At the same photo-irradiation time, chit- osan accelerated rhEGF release due to Pluronic chains (b-PPO-b-PEO-b- PPO) inhibiting the chitosan from being tightly crosslinked because of hydrophobic interactions. Increasing exposure time led to a tight net- work which can control growth factor release (Choi & Yoo, 2010b).

3.1.3. Graft polymerization Graft Polymers are segmented copolymers with a linear backbone made of one polymer and randomly distributed branches made of an- other polymer. Grafting of natural polymers such as chitosan containing two types of reactive groups is of considerable interest for modification of polymer structure. Chitosan has two types of reactive groups that can Fig. 3. Cross linking between Genipin and Chitosan (Muzzarelli et al., 2015). be grafted; the free amine groups on deacetylated units and the

449 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460 hydroxyl groups on the C3 and C6 carbons of acetylated or deacetylated intermolecular and intramolecular hydrogen bonding interaction of units. Recently researchers have shown that chitosan derivatives, such chitosan molecules. Consequently, buffer solution can easily diffuse as hydroxypropyl chitosan (HPCT) and carboxymethyl chitosan sodium into the network and lead to increased swelling ratios. In neutral and (CMCTS) functionalized by grafting, improved water solubility, anti- basic conditions, no protonation occurs and the swelling ratio is low. bacterial, and antioxidant properties (Alves & Manoa, 2008; Xie et al., Mahdavinia et al. (Mahdavinia, Pourjavadi, Hossein zadeh, and 2002; Xie et al., 2001). Zohuriaan, (2004)) introduced graft copolymerization of mixtures of acrylic acid (AA) and acrylamide (AAm) onto chitosan by using PPS as a 3.1.3.1. Chemical grafting. Chemical grafting is initiated by generating free radical initiator and methylene bis-acrylamide (MBA) as a cross one or more free radicals on the chitosan chain and allowing these linker. It was concluded that the swelling capacity of the hydrogels is radicals to react with polymerizable monomers that build the grafted affected by the MBA cross linker concentration and monomer ratio, so chain (Girin et al., 2012). In recent years, a number of initiators such as that the swelling decreases with increased MBA concentration and Ferrous Ammonium Sulfate (FAS), Potassium PerSulfate (PPS), Ceric AAm/AA ratio. They also concluded that this pH–sensitive super- Ammonium Nitrate (CAN), and Ammonium PerSulfate (APS) have been absorbent poly-ampholytic network may be considered as an excellent developed to initiate grafting copolymerization. candidate to design influential drug delivery systems. Yazdani-Pedram et al. (Yazdani-Pedram, Retuert, and Quijada, (2000)) modified chitosan by grafting with polyacrylic acid, a well- 3.1.3.2. Radiation grafting. Grafting can also be initiated by the use of known hydrogel forming polymer. The reaction was carried out in a high energy radiation from gamma rays and from electron beams. homogeneous aqueous phase by using PPS and FAS as the redox in- Radiation graft copolymerization is a well-established technique for itiators. It was observed that the efficiency of grafting depended on producing polymeric materials which combine chemical and physical monomer, initiator, and ferrous ion concentrations, as well as reaction properties of both base polymer and grafted monomer. Grafting time and temperature. Also the level of grafting could be controlled by copolymerization of thermal and pH-sensitive chitosan and N- varying the amount of ferrous ions used as a co-catalyst in the reaction. isopropyl acrylamide hydrogels were investigated by γ-radiation (Cai Jung et al. (Jung, Chung, and Lee, (2006)) prepared a chitosan and et al., 2005). In this research the effects of monomer concentration and eugenol hydrogel to enhance and maintain antioxidant activities by irradiation dose on grafting percentage and efficiency were examined. using CAN as initiator. Eugenol is generally recognized as a safe ma- Grafting percentage and efficiency increased with increased monomer terial by the Food and Drug Administration, so it can be used in bio- concentration and total irradiation dose. However, γ-radiation degrades logical applications. chitosan. Sterilization of chitosan by γ-radiation leads to discoloration The graft yield increased with the concentration of CAN. Thermal and loss of amino groups and lowering of molecular weight. stabilities of chitosan alone and eugenol-chitosan hydrogels were Compared to other methods, radiation crosslinking doesn’t require measured using TGA analysis and it was observed that the eugenol- any additives to start the process, so the final product contains only chitosan hydrogels have a faster thermal decomposition in comparison polymer. Moreover, ionizing radiation usually provides combination of with that of chitosan alone. This is likely because the introduction of the synthesis and sterilization of polymeric materials in a single tech- the bulky eugenol side chains inside the hydrogel decreased the crys- nological step, which leads to reduced cost and production time. talline regions of chitosan. They observed that the equilibrium–swelling Therefore, ionizing radiation or electron beam radiation methods are an ratio of eugenol-grafted chitosan hydrogels is smaller than that of excellent tool in the fabrication of materials for biomedical applications chitosan gels and decreased with increased graft yield, because of the (Zhao & Mitomo, 2008). A series of hydrogels were prepared from hydrophobicity of the attached eugenol molecules. Because chitosan is polyvinyl alcohol (PVA) and carboxy methylated chitosan with electron a cationic polymer, the swelling ratio of chitosan hydrogels decreased beam irradiation at room temperature. The mechanical properties and with increasing pH values, but the swelling ratios of the more hydro- degree of swelling improved substantially after adding chitosan into the phobic eugenol-grafted chitosan hydrogels were not affected by the pH PVA hydrogels. These blend hydrogels, even at low concentration of of the media, because chitosan amino groups, which were pH sensitive, chitosan (3 wt%), exhibited satisfactory antibacterial activity against E. were grafted with eugenol. coli (Zhao, 2003). In another study, N-maleoyl-chitosan was synthesized The pH responsive property of chitosan hydrogels can be explained by reaction of chitosan with maleic anhydride (MAH) in N,N-dimethyl as indicated in Fig. 4 (Zou et al., 2015). At low pH, the amide groups on formamide (DMF) via electron beam irradiation. Grafting yield and the chitosan can become protonated and the resulting electrostatic re- grafting efficiency increased with increasing absorbed radiation dose pulsion between the protonated amino groups weaken the and monomer amount, and then decreased. Usually, the free radicals in

Fig. 4. Mechanism of pH-responsive swelling behavior of chitosan (Zou et al., 2015).

450 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Fig. 5. Scheme of polyelectrolyte complexing reaction between chitosan and pectin (Archana, Dutta et al., 2013). the reaction system increase with dose and then eventually decay. The and the chemical structure of the polymer (Hoare & Kohane, 2008). swelling ratio of the copolymer hydrogel was low at pH 4–5(Fan et al., Tien et al. (Tiena et al., 2003) studied the N-acylation of chitosan 2009). with fatty acyl chlorides to introduce hydrophobicity for use as a matrix for drug delivery. They concluded that hydrophobic interactions en- 3.2. Physical Cross linking (reversible hydrogels) hance the stability of substituted chitosan via hydrophobic self-as- sembly. Also release of drugs depended on the acyl chain length and the ff Physically cross linked gels have attracted much interest recently degree of acylation and could be managed by di usion, or by swelling ff –β due to the absence of chemical crosslinking agents and reagents. Many followed by di usion. An injectable in situ thermosensitive chitosan - e chemical crosslinking agents are toxic compounds which can be de- glycerophosphate (C GP) gel formulation has been proposed for tissue tached or isolated frequently from prepared gels before application. repair and drug delivery (Ruel-Gariépya et al., 2002). The sol/gel They can also affect the nature of the substances when entrapped (e.g. transition can be summarized as a competition between several inter- proteins, drugs, and cells) (Kamoun et al., 2015). The main dis- molecular interactions: 1) the electrostatic repulsion between positively ff advantages of physical gels are their instability (uncontrolled dissolu- charged chitosan molecules, 2) a screening e ect on this repulsion, β tion may occur), low mechanical resistance, and difficult control of pore induced by the negatively charged -glycerophosphate, and 3) attrac- size (Croisier & Jérôme, 2013). Also free chain ends or chain loops are tive interactions due to hydrophobicity and hydrogen bonding between also present as transient network defects in physical gels (Huffman, the chitosan molecules (Supper et al., 2013). This system can release 2012). Some of the most common physical cross-linking methods for macromolecules over a period of several hours to a few days. The re- – – chitosan hydrogel preparation are now described. sults presented in this work indicated that the liposome C GP system could be used for the delivery of small molecular weight hydrophilic compounds. The release profile of the incorporated compound can be 3.2.1. Hydrophobic interaction controlled by adjusting liposome characteristics, such as size and Polymers with hydrophobic domains can crosslink in aqueous en- composition. This formulation can be a good candidate for local release vironments via reverse thermal gelation, also known as ‘sol-gel’ chem- of drugs and biomedical applications which require the local and con- istry. The hydrophobic segment is coupled to a hydrophilic polymer trolled delivery of pharmaceuticals, such as growth factors in tissue segment by post-polymerization grafting or by directly synthesizing a repair. block copolymer to create a polymer amphiphile. These amphiphiles are water soluble at low temperature. But by increasing the tempera- ture, hydrophobic domains aggregate to minimize the hydrophobic 3.2.2. Polyelectrolyte complexation surface area contacting the bulk water, reducing the amount of struc- Formation of chitosan hydrogels by polyelectrolyte complexation tured water surrounding the hydrophobic domains, and maximizing the (PEC) has become an alternative for cross-linking. In the last decade solvent entropy. The temperature at which gelation occurs depends on there has been an increasing interest in the use of PEC gels formed by the concentration of the polymer, the length of the hydrophobic block, chitosan and polyanions as carriers for drug delivery systems. PECs are

451 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460 generally biocompatible networks exhibiting interesting swelling the hydrogels, because of the space left from the melting ice crystals characteristics. PEC gels formed by the electrostatic attractions between during the thawing stages (Guan et al., 2014). two oppositely charged polyelectrolytes in aqueous solution are known The size of ice crystals formed in the structure increases with the to exhibit unique physical and chemical properties. For instance, the number of repeated freeze/thaw cycles (Guan et al., 2014). Ricciardi electrostatic interactions within the PEC gels are considerably stronger et.al (Ricciardi et al., 2004) studied the structure of physical PVA hy- than most secondary binding interactions. The electrostatic attraction drogels prepared by freeze-thaw cycling with a systematic X-ray between the cationic amino groups of chitosan and the anionic groups powder diffraction technique, as a function of the number of cycles and of the other polyelectrolyte is the main interaction leading to the for- aging time. Their results showed that both the degree of crystallinity of mation of PECs (Argin-Soysal et al., 2009; Berger, Reist, Mayer, Felt, the gels and the size of PVA crystallites increased with increasing Gurny et al., 2004). However, the main drawback of these systems is number of freeze/thaw cycles as illustrated in Fig. 7. The increased their difficult preparation, especially in large-scale processes (Berger, degree of crystallinity is mostly due to the increase in the crystallite Reist, Mayer, Felt, Gurny et al., 2004; Long & Luyen, 1996). size, although formation of new crystalline aggregates could not be Archana et al. (Archana, Dutta, and Dutta, (2013)) have studied a excluded. ternary nano-dressing consisting of titanium dioxide (TiO2) nano- Guanghua et al. (Guanghua et al. (2008)) prepared physically cross- particles loaded into a chitosan pectin polyelectrolyte complex that was linked PVA/chitosan hydrogels by the freeze/thaw technique. The prepared to evaluate biocompatibility, and antimicrobial and in vivo swelling results indicated that the hydrogels have good pH-sensitive wound healing properties. The electrostatic attractions between the properties in acidic environments and maximum swelling rate rises ionized amino groups of chitosan (NH2) and the ionized carboxyl acid with the increase of chitosan content in the blend, but leads to more − groups (COO ) of pectin are the main interactions leading to the for- dissolution at pH 1.0. Longer freeze/thaw cycle times result in a lower mation of the pectin/chitosan PECs illustrated in Fig. 5. The tensile swelling rate. They concluded that the swelling behavior of the com- strength of the blended dressing material increased with decreasing posite hydrogels could be adjusted by changing the chitosan contents pectin content (1:1) and incorporation of TiO2 nanoparticles. This nano- and the freezing/thawing cycle times. dressing exhibited superior antimicrobial activity against five patho- PVA hydrogels including chitosan were synthesized by combined gens (E. coli, S. aereus, P. aeruginosa, B. subtilis and A. niger) and good irradiation and freeze-thaw cycling and their properties were compared blood-compatible properties. The chitosan–pectin–TiO2 dressing mate- with those prepared by irradiation or freeze-thaw cycling alone. It was rial could control evaporative water loss from wound beds at an optimal found that hydrogels made by irradiation alone have large swelling level and absorb more exudates, keeping the wound beds moist without capacity and are transparent, but too weak to be used as a wound risking dehydration or accumulation of exudates. Also the nano-TiO2 dressing. Hydrogels made by irradiation followed by freeze-thaw have particles in the chitosan pectin matrix have been shown to decrease large swelling capacity, good thermal stability, high mechanical cytotoxicity. strength, and translucent appearance. All the hydrogels containing Structurally stable chitosan and γ-poly glutamic acid (γ-PGA) PEC chitosan showed pH sensitivity and antibacterial activity (Yang, Liu hydrogel formed through simple ionic complex interaction, between the et al., 2008). amine groups of chitosan and the carboxylic acid groups of γ-PGA, was Fig. 8 shows the effects of the hydrogel preparation method on confirmed by FTIR spectroscopy (Tsao et al., 2010).. It was found that mechanical compression ratio (Afshari et al., 2015). The mechanical the chitosan–γ-PGA PEC hydrogels show antibacterial activity and strengths of the hydrogels prepared by the combinational method are promoted cell proliferation. Therefore, the chitosan–γ-PGA polyelec- higher than that of the hydrogels prepared only by radiation. This is due trolyte hydrogel appears to have potential as a new material for bio- to the presence of physical crosslinks in addition to chemical crosslinks. medical applications. This fact suggests that in order to increase the mechanical strength of hydrogels prepared by radiation, the freeze-thawing treatment after 3.2.3. Freeze-Thaw processing irradiation is very effective [Yang et al., 2008, 2010; Boynueğri et al., The mechanical properties of chitosan hydrogels are not adequate 2009]. for some biomedical applications and many researchers have tried to improve them. In order to achieve good mechanical and biological 4. Clinical studies performance, a combination of a synthetic and a natural polymer seems to be a good choice. These materials are known in the literature as There are few prospective clinical trials in the area of chitosan hy- ‘bioartificial’ polymeric materials. Among synthetic polymers, poly- drogels for wound repair. Some clinical studies have been reported for vinyl alcohol (PVA) has been widely used in biomedical and bio- using chitosan in various medical fields [Diamond et al., 2003; Méthot chemical applications because of its excellent chemical and physical et al., 2016; Mason & Clarke, 2015]. A research has been done on a properties, biodegradability, easy preparation and low cost. Blends of chitosan gelling fiber dressing (Kytocel®) in Staffordshire community chitosan with PVA have been reported to have good mechanical and care to examine improved healing of patients with chronic non-healing chemical properties (Milosavljević et al., 2010). wounds. KytoCel® (Aspen Medical) is a highly absorbent dressing Hydrogels prepared by freeze – thaw processing of poly vinyl al- composed of chitosan fibers which bond with wound exudate to form a cohol (PVA) solutions have drawn extensive attention. Some ad- clear gel absorbing pathogens and is conformable to the wound bed. vantages of physically cross-linked hydrogels obtained by freeze Over a 4-week period or until complete wound healing, leaking, stri- thawing are the nontoxic, noncarcinogenic, and biocompatibility of the kethrough, pain, time, and malodour were examined. This new ad- resulting polymers, good mechanical strength and the absence of vanced wound dressing has the ability to gel when in contact with crosslinking agents or initiators in the hydrogel synthesis (Ricciardi wound fluid, making it less painful to remove, suitable for moderate to et al., 2004; Smith et al., 2009; Xiaomin et al., 2008). During the high exudate, reduced bioburden, and maintain haemostasis (Mason & freezing step, a liquid–liquid phase separation and formation of ice Clarke, 2015). Another clinical study was conducted at three medical crystals in the polymer-poor phase occurs. Polymer chains in the centers in China. A total of 90 patients with unhealed chronic wounds polymer-rich phase lead to the formation of hydrogen bonding and PVA including pressure ulcers, vascular ulcers, diabetic foot ulcers, and crystallites (See Fig. 6). In addition, the thawing procedure facilitates wounds with minor infections, or at risk of infection, were treated with the interactions and formation of crystalline regions between the re- chitosan wound dressing or traditional Vaseline gauze as a control maining polymers, leading to formation of hydrogel networks (Foshan United Medical Technologies Ltd, Guangdong, China). After 4 (Holloway et al., 2013; Zhang et al., 2013). The ice crystals act as cross- weeks of treatment, the wound area reduction was significantly greater linkers during the formation of hydrogels and leave porous structures in in the test group than the control group. The average pain level in the

452 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Fig. 6. Schematic diagrams showing the mechanisms for hydrogel formation from PVA solutions by the freezing–thawing method (Afshari et al., 2015).

chitosan facilitated wound re-epithelialisation and the regeneration of nerves within a vascular dermis. In addition, digital color separation analysis of donor site scars demonstrated an earlier return to normal skin color at chitosan-treated areas (Stone et al., 2000). Chitosan pre- pared from natural biopolymer chitin and cast into membranes has been tested as wound dressing at the skin‐graft donor site in patients. Bactigras, a commonly used impregnated tulle gras bandage, served as a control. The progress in wound healing was compared by clinical and histological examination. After 10 days, the chitosan‐dressed area had been healed more promptly as compared with the Bactigras dressed area. Moreover, the chitosan membrane showed a positive effect on the re‐epithelialization and the regeneration of the granular layer. The data confirm that chitosan membrane is a potential substitute for human wound dressings (Azad et al., 2004).

Fig. 7. Apparent dimensions of PVA hydrogel crystallites as a function of 5. Chitosan application for drug delivery in wound dressings freeze/thaw cycles (Ricciardi et al., 2004). Wound healing is a complicated process including four overlapping phases: coagulation, inflammation, migration-proliferation (including matrix deposition), and remodeling (Falanga, 2005; Kirsner & Eaglstein, 1993; Velnar et al., 2009). The human body can provide all the requirements for the healing process in normal wounds, unless there is any kind of deficiency of skin function or massive fluid losses in large wounds. However, providing appropriate pH, moisture, oxygen pres- sure, and preventing microbial invasion can accelerate the wound healing process (Field & Kerstein, 1994; Guo & DiPietro, 2010). Wound care for non-healing wounds has recently been a growing concern of many wound dressing applications involving various mechanisms, such as coagulation, inflammation, angiogenesis, epithelization, contraction and remodeling. An ideal wound dressing should protect the wound from bacterial infection, provide a moist and healing environment, and be biocompatible (Boateng, Matthews, Stevens, & Eccleston, 2008; Bhuvanesh, 2010; Paul & Sharma, 2004). Because of chitosan’s pre- viously mentioned significant characteristics, such as biocompatibility, biodegradability, hemostatic and anti-infection activity, and the ability to accelerate wound healing, it has attracted many researchers to be used as a wound dressing material. Applications of chitosan in different types of wound dressing are explained in the following sections.

Fig. 8. Comparison of mechanical strength of hydrogels prepared by 5.1. Application of growth factors in chitosan-based wound dressings Combinational and radiation method (Yang, Zhu et al., 2008).

Growth factors are polypeptides which control the growth, differ- test group was 1.12 ± 0.23 and 2.30 ± 0.23 in the control group. The entiation, and metabolism of cells and regulate the process of tissue mean duration of the test group was 27.31 ± 5.37 days and the control repair. Despite their small amounts, they exert a powerful influence on group 27.09 ± 6.44 days. Consequently, the new chitosan wound the process of wound repair. Use of growth factors for accelerating the dressing can enhance the wound healing process and reduce the pa- healing of wounds presents tremendous promise as a therapeutic ap- ’ tient s pain level. Furthermore the dressing was shown to be clinically proach in treating chronic wounds. Several peptide growth factors, such ff safe and e ective in the management of chronic wounds (Mo et al., as epidermal growth factor (EGF), platelet derived growth factor 2015). (PDGF), fibroblast growth factor (FGF) and transforming growth factor- An evaluation of healing at split skin graft donor sites, dressed half beta (TGF-β), have been shown to accelerate cellular proliferation and with chitosan and half with a conventional dressing, revealed that synthesis of the extracellular matrix (Alemdaroğlu et al., 2006; Paul &

453 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Table 1 5.2. Nanoparticle and nanostructure incorporated hydrogels The average stained cell number in Chitosan gel with and without EGF (Yenilmez et al., 2015). Nanotechnology is developing as a new growing field with appli- Groups The average stained cell number cations in Science and Technology for the purpose of manufacturing new materials at the nanoscale level. Nano-particles are used not only 3rd day 7th day 14th day in fundamental chemistry and physics investigations, but also widely in * J 1.20 ± 0.17 2.43 ± 0.61 3.10 ± 0.68 biomedical fields. Due to their good antibacterial properties, their large EJ* 1.63 ± 0.35 4.20 ± 0.18 4.95 ± 0.39 surface area to volume ratios, and high microbial resistance, nanoscale * Burn wounds treated by chitosan gel without (J) and with (EJ) with EGF. materials have emerged as novel antimicrobial agents (Sung et al., 2010; Radhakumary et al., 2011). Different types of metal materials Sharma, 2004). EGF has been reported to increase the rates of cellular have been known to be used as antibacterial and/or/ antimicrobial proliferation, growth and regeneration of tissue in numerous papers agents in wound healing such as, silver, zinc oxide, titanium dioxide, (Choi et al., 2012; Su et al., 2014; Yenilmez et al., 2015). copper oxide, and graphene. However, there could be problems with Regarding EGF and chitosan properties for wound healing, long term exposure. Alemdaroğlu et al. (Alemdaroğlu et al. (2006)) developed an effective Sudheesh Kumar et al. (Kumbar et al., 2003) developed a flexible chitosan hydrogel formulation containing EGF to determine its effect on and microporous chitosan/nano zinc oxide composite hydrogel. At healing of second-degree burn wounds in rats. Burns are classified ac- neutral pH, chitosan does not show much antibacterial activity; in order cording to the depth of the injury. In superficial second-degree burns, to impart that activity, it is necessary to incorporate some antibacterial the epidermis and the superficial dermis are mainly affected. And these agents into it. Zinc oxide nanoparticles were used in this research due to kinds of burns are very painful. According to the in vitro release studies, its antibacterial activity. in vitro cytocompatibility studies revealed that the release of EGF from the chitosan gel was found to be 97.3% after the dressing showed enhanced cell viability and infiltration. in vivo 24 h. The release kinetics from the gel formulation was found to be of wound healing evaluation proved the healing ability and antibacterial the first order, and the release rate of EGF from the gel varied with time. potential of the prepared hydrogel without causing toxicity to cells. The in vivo study exhibited significant increase in cell proliferation in They found that these advanced dressings can be used to prevent in- the EGF containing gel applied group. The average numbers of pro- fections in burns and chronic and diabetic wounds. It is also possible to liferating cells observed on the 3rd, 7th and 14th days are presented in synthesize chitosan quaternary salts that have a permanent cationic Table 1. Evaluation of these immunehisto-chemically observed results site, at all pH. showed that the rate of healing in the EJ group was increased. Finally it Silver and silver ions have been known as effective antimicrobial has concluded that this formulation can play an important role in burn agents for a long time. Owing to this ability, they have been applied to a wound treatment. wide range of healthcare products, such as burn dressings, scaffolds, Park et al. (Park, Clark, Lichtensteiger, Jamison, and Wagoner skin replacements and medical devices (Altiok et al., 2010). Johnson, (2009)) prepared chitosan scaffolds loaded with basic fibro- Nano silver/gelatin/carboxymethyl (CM)-Chitosan hydrogels were blast growth factor (bFGF) contained in gelatin micro-particles and synthesized by radiation-induced reduction and crosslinking at ambient tested for clinical relevance in an aged mouse model. The application of temperature (Altiok et al., 2010). After incubation at 37 °C for 12 h, this kind of wound dressing was for accelerating the healing process of these hydrogels clearly showed antibacterial activity against gram-ne- pressure ulcers. bFGF was successfully delivered to the wound and in- gative E. coli. The hydrogel with 10 mM nano silver content inhibited creased angiogenesis compared to chitosan alone. They showed that more than 99% of the E. coli. When the nano silver content decreased to levels of bFGF in wound tissue were significantly higher, indicating 5 mM, the maximum inhibition ratio was around 50%, and when the successful and prolonged growth factor delivery without burst effects. nano silver content was further decreased to 2 mM, the ratio decreased Finally they concluded that chitosan loaded with bFGF is a good can- to 26%. The results of this study suggest that nano silver/gelatin/CM- didate for treatment of chronic wounds and reduces the proteolytic Chitosan hydrogels have potential as an antibacterial and anti-in- environment of the wound and potentiates bFGF activity, leading to flammation wound dressing. accelerated wound closure. Nano-curcumin was incorporated in N,O-carboxymethyl chitosan/ Table 2 Summarizes some reported chitosan hydrogels with in- oxidized alginate hydrogel to develop a novel nano-composite hydrogel corporated growth factors for wound dressing applications. wound dressing (Gaysinsky et al., 2008). Curcumin, a natural product of the rhizomes of Curcuma longa, has been widely used as coloring

Table 2 Applications of incorporated growth factors in chitosan hydrogels.

Growth Factors Preparation Technique Potential Application References

EGF* Mixing chitosan gel with GF Treating second-degree burn wounds (Yenilmez et al., 2015) EGF Semi-IPN hydrogel Wound dressing (Morones et al., 2005) EGF-liposome – Treating second-degree burn wounds (Sudheesh Kumar et al., 2012) EGF & VEGF* Ionotropic gelation Wound healing accelerator (Zhou et al., 2012) rh-EGF* Photo crosslinking Diabetic ulcers wound dressing (Li, Chen et al., 2012) FGF-1 & FGF-2* Photo crosslinking Carrier material & Wound healing accelerator (Mani et al., 2002) FGF-2* Photo crosslinking Treating healing-impaired wounds (Obara et al., 2003) b FGF – Treating pressure ulcers (Rai et al., 2009) b FGF Polyelectrolyte complexes (PEC) Skin tissue regeneration (Nimal et al., 2016) PDGF-BB* & VEGF* Visible light activated crosslinking Wound healing accelerator (Bhattarai et al., 2010)

* EGF: Epidermal growth factor, VEGF: Vascular endothelial growth factor, rh-EGF: recombinant human epidermal growth factor, FGF-1: fibroblast growth factor- 1, PDGF-BB: Platelet derived growth factor-BB.

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Table 3 Application of nanoparticles Chitosan hydrogels.

Drug Preparation Technique Potential Application References

Zinc oxide / Silver Genipin-crosslinking Burn dressings (Yang et al., 2017) Silver sulfadiazine Genipin-crosslinking As a carrier dressing (Choi & Yoo, 2010a) Silver Oxide Casting/solvent evaporation Wound dressing (Pulat et al., 2013) Silver UV radiation Biomedical fields (Ishihara et al., 2003) UV radiation Anti-infectious wound dressing (Ho et al., 2009) Glutaraldehyde-crosslinking Anti-microbial wound dressing (Liu & Kim, 2012a) Copper oxide Epichlorohydrin-crosslinking Biomedical fields (Tyliszczak et al., 2017) Zinc oxide Casting/solvent evaporation Wound dressing (Hajji et al., 2017) Zinc oxide Genipin-crosslinking Burn dressings (Wahid et al., 2017) Titanium dioxide Casting/solvent evaporation Wound healing dressing (Değim et al., 2011) Graphene oxide Schiff-base reaction* Wound dressing (Vasile et al., 2014) Curcumin nanoformulation Casting/solvent evaporation Wound healing dressing (Archana et al., 2015) Nano Curcumin Mixing wound dressing (Gaysinsky et al., 2008) Tigecycline nanoparticles Mixing Infectious wound treatment (Galotto et al., 2016)

*Reaction between aldehyde and the amino group of two materials. agent and spice in food. But studies have shown that curcumin could interaction of chitosan with PVA. According to their histologicaly ex- significantly accelerate the healing of wounds and enhance wound re- amined data presented in Table 4, hydrogels prepared with drug re- pair in diabetic impaired healing (Gaysinsky, 2007). Nano-curcumin sulted in less inflammatory cells, more numerous collagen prolifera- could be released slowly from hydrogel to stimulate fibroblast pro- tions, and microvessels in rats than the conventional product or the liferation, capillary formation and collagen production which sig- control (sterile gauze). The healing effect of minocycline-loaded hy- nificantly has effect on the healing phases. So, the nano-curcumin/ drogel was great, indicating the potential healing effect of minocycline. chitosan/alginate hydrogel might have potential application in wound Finally they concluded that the aforementioned minocycline-loaded healing. wound dressing is a potential wound dressing with excellent forming Tigecycline nanoparticles were loaded into chitosan–platelet-rich and enhanced wound healing characteristics. plasma hydrogel for wound infection treatment. The tigecycline nano- A thermo responsive and cytocompatible chitosan based hydrogel particle incorporated chitosan hydrogel showed a significant anti- consisting of thiolated chitosan with poly (N-isopropyl acrylamide) bacterial activity against S. aureus. This system can be an effective loaded with ciprofloxacin was introduced by Radhakumary et al medium for antibiotic delivery and applied on the infection sites to (Mishra et al., 2017). They reported that polymers with thiol groups effectively prevent various skin infections caused by S. aureus (Galotto provide enhanced adhesive properties in comparison with many mu- et al., 2016). coadhesive polymers, and the strong cohesive properties of thiolated Table 3 Summarizes some reported Chitosan hydrogels containing chitosan make it highly suitable for controlled drug release. Cipro- nanoparticles used for wound dressing applications. fl oxacin is a quinolone antibiotic drug recognized for treatment of skin and skin structure infections. Their prepared film exhibited appropriate mechanical properties for a wound/burn dressing and was found to be 5.3. Drug incorporated hydrogels cytocompatible and antibacterial as they released the entrapped anti- biotic in a controlled manner for a period of more than 48 h. Controlled delivery systems provide an alternative approach to Hydrogel microspheres of polyacrylamide-grafted‐chitosan crosslinked regulate bioavailability of therapeutic agents. In controlled drug de- with glutaraldehyde were prepared to encapsulate indomethacin, a non- livery systems, an active therapeutic is incorporated into a polymeric steroidal anti‐inflammatory drug. The microspheres were produced by the network structure, and the drug is released from the structure in a water/oil emulsion technique and encapsulation of indomethacin was car- predefined manner. Depending on the drug delivery formulation and ried out before crosslinking of the matrix. The initial release of in- application, the drug release time may be from a few hours to several domethacin is due to the polymer chain relaxation process, but at longer months (Vimala et al., 2011). Different approaches have been devel- times, the release occurs from the fully swollen polymer and is controlled oped for drug incorporation. The method of drugs loading directly mainly by the molecular diffusion phenomenon. This study suggests that impacts the availability of the drugs during release. the hydrogel microspheres prepared from chitosan‐based polymers can be According to the application of wound dressings and the types of useful in the delivery of indomethacin‐like drugs (Zhang et al., 2018). wounds, various kinds of drugs can be loaded in wound dressings. Sung Additional drug-loaded chitosan hydrogels reported for wound et al. (Zhang et al., 2015) developed a minocycline-loaded wound dressing applications are presented in Table 5. dressing made with a PVA and Chitosan blended hydrogel made using Essential oils and herbal extracts are natural complex mixtures of the freeze-thaw method. Minocycline was used in this study because it volatile, lipophilic substances obtained from different parts of plants by has been used in the treatment of superficial infections of the skin. The steam distillation and solvent extraction. In recent decades, interest in minocycline-loaded wound dressing composed of 5% PVA, 0.75% essential oils for use in pharmaceutical and biomedical field has risen. chitosan and 0.25% drug was more swellable, flexible and elastic than Essential oils have been shown to possess antibacterial, antifungal, the PVA gel alone, because of the relatively weak cross-linking

Table 4 Histomorphometrical Values for a Minocycline-loaded wound dressing made with a PVA and Chitosan blended hydrogel (Zhang et al., 2015).

Groups histomorphometry Gauze (control) Conventional product Hydrogel without drug Hydrogel with drug

Numbers of microvessels (vessels/1 mm2 of field) 4.40 ± 1.67 20.80 ± 6.72a 3.20 ± 2.28 23.40 ± 8.99 Numbers of inflammatory cells (cells/1 mm2 of field) 2106.00 ± 934.45 250.00 ± 217.17a 2447.00 ± 527.81 179.00 ± 227.74 Percentages of collagen tissue occupied regions (%/1 mm2 of field) 36.73 ± 8.96 66.21 ± 10.07a 33.41 ± 4.50 75.81 ± 9.44

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Table 5 Application of drug-loaded Chitosan hydrogels.

Drug Preparation Technique Potential Application References

Gentamicin sulfate EDC/NHS*-crosslinking Anti-bactericidal wound dressing (Chen et al., 2017) Gentamicin sulfate EDC/NHS*-crosslinking Anti-bactericidal wound dressing (Patel et al., 2017) Tetracycline hydrochloride Emulsion-crosslinking Anti-bacterial wound dressing (Yang et al., 2018) Tetracycline hydrochloride Mixing Scar preventive wound healing (Fan et al., 2016) Tetracycline hydrochloride / silver sulfadiazine Casting/solvent evaporation Anti-infection wound dressing (Gao et al., 2016) Naproxen Casting/solvent evaporation Anti-infection wound dressing (Ma et al., 2017) Superoxide dismutase Polyelectrolyte complexes Antioxidant wound dressing (Rocasalbas et al., 2013) Lignin Freeze-thaw Wound dressings (Morgado et al., 2017) Amoxicillin Freeze-thaw Antibiotic delivery (Akıncıbay et al., 2007) Ibuprofen Not mentioned Wound dressings (Perchyonok et al., 2014) Metronidazole benzoate Mixing Treatment of chronic periodontitis (Ribeiro et al., 2013) Clarithromycin, Tramadol and Heparin Genipin-crosslinking Pharmaceutical applications (Archana et al., 2013) Nystatin Mixing Wound healing application (Liu & Kim, 2012b) Acidic oxyphenbutazone & glafenine Coacervation techniques Anti-inflammatory drugs (Harris et al., 2008) Essential oils or herbal extracts Nerolidol – Wound healing dressing (Gonçalves Ferreira et al., 2016) Thymol NVP-crosslinking* Wound dressing (Qin et al., 2006) Apigenin PEG-crosslinking* Diabetic wound dressing (Kim et al., 2015) Lupeol Glutaraldehyde-crosslinking Wound dressing (Risbud et al., 2000) Polyphenolic Laccase- crosslinking Chronic wound dressing (Li et al., 2012)

* EDC: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiiminde, NHS: N-hydroxysuccinimide, PEG: Poly ethylene glycol. NVP: 1-vinyl-2-pyrrolidone. antiviral, insecticidal and antioxidant properties due to their biologi- Ahmed, E. M. (2015). Hydrogel: Preparation, characterization, and applications: A re- cally active compounds (See Table 5. for their applications in wound view. Journal of Advanced Research, 6(2), 105–121. fi Ajji, Z., Othman, I., & Rosiak, J. M. (2005). Production of hydrogel wound dressings using dressings). Chitosan lms loaded with thyme oil were prepared by a gamma radiation. NIMB, 229, 375–380. solvent casting method (Jiang et al., 2016). Thyme oil has been shown Akıncıbay, H., Şenel, S., & Yetkin Ay, Z. (2007). Application of chitosan gel in the to have inhibitory activities against various bacteria and yeasts (Shukla treatment of chronic periodontitis. Journal of Biomedical Material Research, Part B: Applied Biomaterials, 80B(2), 290–296. et al., 2016). Thymol, the major component of thyme oil (Anjum et al., Alemdaroğlu, C., Değim, Z., Çelebi, N., Zor, F., Öztürk, S., & Erdoğan, D. (2006). An 2016), has also been shown to exhibit antimicrobial activity against investigation on burn wound healing in rats with chitosan gel formulation containing several bacteria and fungi (Koosehgol et al., 2017). The prepared films epidermal growth factor. Burns, 32(3), 319–327. showed both antibacterial and antioxidant activities. Results of this Altiok, D., Altiok, E., & Tihminlioglu, F. (2010). Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing research revealed that the thyme oil has a good potential to be in- applications. Journal of Materials Science: Materials in Medicine, 21(7), 2227–2236. corporated into chitosan to make antibacterial and permeable films for Alves, N. M., & Manoa, J. F. (2008). Chitosan derivatives obtained by chemical mod- fi wound healing applications. i cations for biomedical and environmental applications. International Journal of Biological Macromolecules, 43, 401–414. Alvesab, A., Pinhoab, E. D., Nevesab, N. M., Sousaab, R. A., & Reisab, R. L. (2012). 6. Conclusions Processing ulvan into 2D structures: Cross-linked ulvan membranes as new bioma- terials for drug delivery applications. International Journal of Pharmaceutics, 426, 76–81. Wound healing is a complex and dynamic process of replacing de- Anjum, S., Arora, A., Alam, M. S., & Gupta, B. (2016). Development of antimicrobial and vitalized and missing cellular structures and tissue layers. Many efforts scar preventive chitosan hydrogel wound dressings. International Journal of have been focused on wound care with an emphasis on new therapeutic Pharmaceutics, 508(1-2), 92–101. Archana, D., Singh, B. K., Dutta, J., & Dutta, P. K. (2015). Chitosan-PVP-nano silver oxide approaches and development of technologies for wound management. wound dressing: In vitro and in vivo evaluation. International Journal of Biological In recent decades, smart wound dressings with accelerated wound Macromolecules, 73,49–57. healing properties have attracted much interest. Archana, D., Singh, B. K., Dutta, J., & Dutta, P. K. (2013). In vivo evaluation of chitosan–PVP–titanium dioxide nanocomposite as wound dressing material. This review summarized the potential applications of chitosan hy- Carbohydrate Polymers, 95(1), 530–539. drogels for pharmaceutical and biomedical uses, particularly with re- Arcidiacono, S., & Kaplan, D. L. (1992). Molecular weight distribution of chitosan isolated gard to drug delivery in wound dressings. Chitosan is an excellent from mucor rouxii under different culture and processing conditions. Biotechnology – candidate due to its non-toxicity, stability, biodegradability, and bio- and Bioengineering, 39(3), 281 286. Argin-Soysal, S., Kofinas, P., & Lo, Y. M. (2009). Effect of complexation conditions on compatibility. Also chitosan hydrogel structures may be loaded with xanthan–chitosan polyelectrolyte complex gels. Food Hydrocolloids, 23(1), 202–209. various types of drugs in different physical forms, such as nanoparticles, Arias, J. L., Neira-Carrillo, A., Arias, J. I., Escobar, C., Bodero, M., David, M., et al. (2004). essential oils, and solubilized drugs. These properties have made chit- Sulfated polymers in biological mineralization: A plausible source for bio-inspired engineering. Journal of Materials Chemistry, 14, 2154–2160. osan a very versatile material with extensive applications in controlled Arrouze, F., Essahli, M., Rhazi, M., desberieres, J., & Tolaimate, A. (2017). Chitin and release formulations for treatment of acute and chronic wounds like chitosan: Study of the possibilities of their production by valorization of the waste of burns and pressure and diabetic ulcers. Therefore it can be concluded crustaceans and cephalopods rejected in Essaouira. Journal of Materials and Environmental Sciences, 8(7), 2251–2258. that chitosan-based hydrogels are expected to become a promising Aspden, T., Illum, L., & Skaugrud, Q. (1997). The effect of chronic nasal application of matrix for use in regenerative medicine, drug delivery, and wound chitosan solution on cilia beat frequency in guinea pigs. Int. J. Pharm, 153, 137–146. dressing applications. Aspden, T., Adler, J., Davis, S. S., Skaugrud, Q., & Illum, L. (1995). Chitosan as a nasal delivery system: Evaluation of the effect of chitosan on mucociliary clearance rate in frog plate model, int. J. Pharm, 122,69–78. References Aspden, T. J., Mason, J. D. T., Jones, N., Lowe, J., Skaugrud, Q., & Illum, L. (1994). Chitosan as a nasal delivery system: The effect of chitosan on invitro and in vivo mucociliary transport rates. J. Pharm. Sci, 86, 509–513. Abd El-Mohdy, H. L. (2013). Radiation synthesis of nanosilver/poly vinyl alcohol/cellu- Aspden, T., Illum, L., & Skaugrud, Q. (1996). Chitosan as anasal delivery system: lose acetate/gelatin hydrogels for wound dressing. Journal of Polymer Research, Evaluation of insulin absorption enhancement and effect on nasal membrane in- 20(177), 1–12. tegrety using rat models. Eur. J. Pharm. Sci, 4,23–31. Afshari, M. J., Sheik, N., & farideh, bH. A. (2015). PVA/CM-chitosan/honey hydrogels AXIO (2018). Axiostat, [online]. Available:http://www.axiobio.com/emergency/. prepared by using the combined technique of irradiation followed by freeze-thawing. Azad, A. K., Sermsintham, N., Chandrkrachang, S., & Stevens, W. F. (2004). Chitosan Radiation Physics and Chemistry, 113,28–35.

456 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

membrane as a wound‐healing dressing: Characterization and clinical application. Ezra, D. G., Chan, M. P. Y., Solebo, L., Malik, A. P., Crane, E., Coombes, A., et al. (2009). Journal of Biomaterial Research. Part B: Applied Bomaterial, 69B(2). Randomised trial comparing ocular lubricants and polyacrylamide hydrogel dressings Balakrishnan, B., Mohanty, M., Umashankar, P. R., & Jayakrishnan, A. (2005). Evaluation in the prevention of exposure keratopathy in the critically ill. Intensive Care Medicine, of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. 35, 455–461. Biomaterials, 26(32), 6335–6342. Falanga, V. (2005). Wound healing and its impairment in the diabetic foot. THE LANCET, Baldrick, P. (2010). The safety of chitosan as a pharmaceutical excipient. Regulatory 366(9498), 1736–1743. Toxicology and Pharmacology, 56, 290–299. Fan, J., Chen, J., Yang, L., Lin, H., & Cao, F. (2009). Preparation of dual-sensitive graft Benamer, S., Mahlous, M., Boukrif, A., Mansouri, B., & Larbi Youcef, S. (2006). Synthesis copolymer hydrogel based on N-maleoyl-chitosan and poly(N-isopropylacrylamide) and characterisation of hydrogels based on poly(vinyl pyrrolidone). Nuclear by electron beam radiation. Bulletin of Materials Science, 32(5), 521–529. Instruments and Methods in Physics Research B, 248, 284–290. Fan, L., Yi, J., Tong, J., Zhou, X., Ge, H., Zou, S., et al. (2016). Preparation and char- Bhattarai, N., Gunn, J., & Zhang, M. (2010). Chitosan-based hydrogels for controlled, acterization of oxidized konjac glucomannan/carboxymethyl chitosan/graphene localized drug delivery. Advanced Drug Delivery Reviews, 62(1), 83–99. oxide hydrogel. International Journal of Biological Macromolecules, 91, 358–367. Bhuvanesh, A. R. M. S. A. G. (2010). Textile-based smart wound dressings. Indian Journal Field, C. K., & Kerstein, M. D. (1994). Overview of wound healing in a moist environment. of Fibre & Textile Research, 35(2), 174–185. American Journal of Surgery, 167(1), S2–S6. Bigi, A., Cojazzi, G., Panzavolta, S., Roveri, N., & Rubini, K. (2003). Stabilization of ge- Gades, M. D., & Stern, J. S. (2003). Chitosan supplementation and fecal fat excretion in latin films by crosslinking with genipin. Biomaterials, 23, 4827–4832. men. Obesity, 11(5), 683–688. -E., et al. (2009). Clinical and Galotto, M. J., de Dicastillo, C. L., Torres, A., & Guarda, A. (2016). Thymol: Use in an ,ﻭBoynueğri, D., Özcan, G., Şenel, S., Uç, D., Uraz, A., Öğüş radiographic evaluations of chitosan gel in periodontal intraosseous defects: A pilot timicrobial packaging. Antimicrobial Food Packaging, 553–562. study. Journal of Biomedical Research Part B: Biomaterials, 90B(1), 461–466. Gao, L., Gan, H., Meng, Z., Gu, R., Wu, Z., Zhang, L., et al. (2014). Effects of genipin cross- Bueter, C. L., Lee, C. K., Rathinam, V. A. K., Healy, G. J., Taron, C. H., Specht, C. A., et al. linking of chitosan hydrogels on cellular adhesion and viability. Colloids and Surfaces (2011). Chitosan but not chitin activates the inflammasome by a mechanism de- B: Biointerfaces, 117, 398–405. pendent upon phagocytosis. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 286, Gao, L., Gan, H., Meng, Z., Gu, R., Wu, Z., Zhu, X., et al. (2016). Evaluation of genipin- 35447–35455. crosslinked chitosan hydrogels as a potential carrier for silver sulfadiazine nano- Cai, J., Yang, J., Du, Y., Fan, L., Qiu, Y., Li, J., et al. (2006). Enzymatic preparation of crystals. Colloids and Surfaces B: Biointerfaces, 148(1), 343–353. chitosan from the waste aspergillus niger mycelium of citric acid production plant. Gaysinsky, S. (2007). Emulsions and micro emulsions as antimicrobial deliverysystems. Carbohydrate Polymers, 64(2), 151–157. Amherst. Cai, H., Zhang, Z. P., Sun, P. C., He, B. L., & Xia Zhu, X. (2005). Synthesis and char- Gaysinsky, S., Davidson, P., McClements, D., & Weiss, J. (2008). Formulation and char- acterization of thermo- and pH- sensitive hydrogels based on chitosan-grafted N- acterization of phyto-phenol-carrying antimicrobial micro emulsions. Food Bio isopropylacrylamide via γ-radiation. Radiation Physics and Chemistry, 74(1), 26–30. Physics, 3(1), 54–65. Caló, E., & Khutoryanskiy, V. V. (2015). Biomedical applications of hydrogels: A review of Girin, T. K., Thakur, A., Alexander, A., Ajazuddin, H., Badwaik, D. K., & Tripathi (2012). patents and commercial products. European Polymer Journal, 65, 252–267. Modified chitosan hydrogels as drug delivery and tissue engineering systems:Present Carreño-Gómez, B., & Duncan, R. (1997). Evaluation of the biological properties of so- status and applications. Acta Pharmaceutica Sinica B, 2(5), 439–449. luble chitosan and chitosan microspheres. International Journal of Pharmaceutics, Gonçalves Ferreira, M. O., Ribeiro Leite, L. L., Lima, I. S., Medeiros Barreto, H., Cunha 148(2), 231–240. Nunes, L. C., Ribeiro, A. B., et al. (2016). Chitosan hydrogel in combination with Chang, C., Duan, B., & Zhang, L. (2009). Fabrication and characterization of novel nerolidol for healing wounds. Carbohydrate Polymers, 152, 409–418. macroporous cellulose–alginate hydrogels. Polymer, 50(23), 5467–5473. Guan, Y., Bian, J., Peng, F., Zhang, X. M., & Sun, R. C. (2014). High strength of hemi- Chang, W. H., Chang, Y., Chen, Y. C., & Sung, H. W. (2004). Hemoglobin Polymerized celluloses based hydrogels by freeze/thaw technique. Carbohydrate Polymers, 101, with a Naturally Occurring Crosslinking Agent as a Blood Substitute: In Vitro and In 272–280. Vivo Studies. Artificial Cells, Blood Substitutes, and Biotechnology, 32(2), 243–262. Guanghua, H., Hua, Z., & Fuliang, X. (2008). Preparation and swelling behavior of Chen, R. H., Chang, J. R., & Shyur, J. S. (1997). Effects of ultrasonic conditions and physically crosslinked hydrogels composed of poly(vinyl alcohol) and chitosan. storage in acidic solutions on changes in molecular weight and polydispersity of Journal of Wuhan University of Technology-Mater, 23(6). treated chitosan. Carbohydrate Research, 299(4), 287–294. Guo, S., & DiPietro, L. A. (2010). Factors affecting wound healing. Critical Reviews in Oral Chen, S. C., Wu, Y. C., Mi, F. L., Lin, Y. H., Yu, L. C., & Sung, H. W. (2004). A novel pH- Biology & Medicine, 89(3), 219–229. sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross- Gupta, B., Saxena, S., Arora, A., & Alam, M. S. (2011). Chitosan-polyethylene glycol linked by genipin for protein drug delivery. Journal of Controlled Release, 96(2), coated cotton membranes for wound dressings. Indian Journal of Fiber & Textile 258–300. Research, 36, 272–280. Chen, H., Xing, X., Tan, H., Jia, Y., Zhou, T., Chen, Y., et al. (2017). Covalently anti- H.M.T. Inc (2007). ChitoFlex. Patent reg. No. 3264070. 17 July. bacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres con- Hajji, S., Slama-Ben Salem, R. B., Hamdi, M., Jellouli, K., Ayadi, W., Nasri, M., et al. taining tetracycline hydrochloride for wound healing. Materials Science and (2017). Nanocomposite films based on chitosan–poly(vinyl alcohol) and silver na- Engineering: C, 70(1), 287–295. noparticles with high antibacterial and antioxidant activities. Process Safety and Choi, J. S., & Yoo, H. S. (2010a). Pluronic/chitosan hydrogels containing epidermal Environmental Protection, 111, 112–121. growth factor with wound-adhesive and photo-crosslinkable properties. Journal of Harris, R., Lecumberri, E., & Heras, A. (2008). Chitosan-genipin microspheres for the Biomedical Material Research, 95A(2), 564–573. controlled release of drugs: Clarithromycin, tramadol and heparin. Marine Drugs, Choi, J. S., & Yoo, H. S. (2010b). Pluronic/chitosan hydrogels containing epidermal 8(6), 1750–1762. growth factor with wound-adhesive and photo-crosslinkable properties. Journal Og Helander, I. M., Nurmiaho-Lassila, E. L., Ahvenainen, R., Rhoades, J., & Roller, S. (2001). Biomedical Material Research, 95A(2), 564–573. Chitosan disrupts the barrier properties of the outer membrane of gram-negative Choi, J. K., Jang, J. H., Jang, W. H., Kim, J., Bae, I. H., Bae, J., et al. (2012). The effect of bacteria. International Journal of Food Microbiology, 71, 235–244. epidermal growth factor (EGF) conjugated with low-molecular-weight protamine Hennink, W. E., & Nostrum, C. F. (2002). Novel crosslinking methods to design hydrogels. (LMWP) on wound healing of the skin. Biomaterials, 33, 8579–8590. Advanced Drug Delivery Reviews, 54(1), 13–36. Chung, L. Y., Schmidt, R. J., Hamlyn, P. F., Sagar, B. F., Andrews, A. M., & Turner, T. D. Ho, Y., Mi, F., Sung, H., & Kuo, P. (2009). Heparin-functionalized chitosan–alginate (1994). Biocompatibility of potential wound management products: Fungal mycelia scaffolds for controlled release of growth factor. International Journal of as a source of chitin/chitosan and their effect on the proliferation of human f1000 Pharmaceutics, 376(1-2), 69–75. fibroblasts cultur. J of Biomecical Materials Research, 28, 463–469. Hoare, T. R., & Kohane, D. S. (2008). Hydrogels in drug delivery: Progress and challenges. Chung, Y., Su, Y., Chen, C., Jia, G., Wang, H., Wu, J. G., et al. (2004). Relationship Polymer, 49(8), 1993–2007. between antibacterial activity of chitosan and surface characteristics of cell wall. Acta Holloway, J. L., Lowman, A. M., & Palmese, G. R. (2013). The role of crystallization and Pharmecutical Sceince, 25(7), 932–936. phase separation in the formation of physically cross-linked PVA hydrogels. Soft Costa, E. M., Silva, S., Costa, M. R., Pereira, M., Campos, D. A., Odila, J., et al. (2014). Matter, 9, 826–833. Chitosan mouthwash: Toxicity and in vivo validation. Carbohydrate Polymers, 111, Horio, T., Ishihara, M., Fujita, M., Kishimoto, S., Kanatani, Y., Ishizuka, T., et al. (2010). 385–392. Effect of photocrosslinkable chitosan hydrogel and its sponges to stop bleeding in a Croisier, F., & Jérôme, C. (2013). Chitosan-based biomaterials for tissue engineering. rat liver injury model. Artificial Organs, 34(4), 342–347. European Polymer Journal, 49(4), 780–792. Hu, X., & Gao, C. (2008). Photoinitiating polymerization to prepare biocompatible chit- Cui, L., Jia, J., Guo, Y., Liu, Y., & Zhu, P. (2014). Preparation and characterization of IPN osan hydrogels. Journal of Applied Polymer Science, 110(2), 1059–1067. hydrogels composed of chitosan and gelatin cross-linked by genipin. Carbohydrate Huffman, A. S. (2012). Hydrogels for biomedical applications. Advanced Drug Delivery Polymers, 99,31–38. Reviews, 64,18–23. Değim, Z., Çelebi, N., Alemdaroğlu, C., Deveci, M., Öztürk, S., & Özoğul, C. (2011). Ik, S. C., Cho, M. O., Li, Z., Nurunnabi, M., Park, S. Y., Kang, S., et al. (2016). Synthesis Evaluation of chitosan gel containing liposome-loaded epidermal growth factor on and characterization of a new photo-crosslinkable glycol chitosan thermogel for burn wound healing. International Wound Journal, 8(4), 343–354. biomedical applications. Carbohydrate Polymers, 144,59–67. Delmar, K., & Bianco-Peled, H. (2015). The dramatic effect of small pH changes on the Ishihara, M., Obara, K., Ishizuka, T., Fujita, M., Sato, M., Masuoka, K., et al. (2003). properties of chitosan hydrogels crosslinked with genipin. Carbohydrate Polymers, Controlled release of fibroblast growth factors and heparin from photocrosslinked 127,28–37. chitosan hydrogels and subsequent effect on in vivo vascularization. Journal of Diamond, M. P., Luciano, A., Johns, D. A., Dunn, R., Young, P., & Bieber, E. (2003). Biomedical Materials Research, 64A(3), 551–559. Eduction of postoperative adhesions by N,O-carboxymethylchitosan: A pilot study. Islam, A., & Yasin, T. (2012). Controlled delivery of drug from pH sensitive chitosan/poly Fertility and Sterility, 80(3), 631–636. (vinyl alcohol) blend. Carbohydrate Polymers, 88(no. 3), 1055–1060. Dowling, M. B., Kumar, R., Keibler, M. A., Hess, J. R., Bochicchio, G. V., & Raghavan, S. R. J.P, C., & T.H, C. (2006). Thermo-responsive chitosan-graft-poly(N-isopropylacrylamide) (2011). A self-assembling hydrophobically modified chitosan capable of reversible injectable hydrogel for cultivation of chondrocytes and meniscus cells. hemostatic action. Biomaterials, 32(13), 3351–3357. Macromolecular Bioscience, 6, 1026–1039.

457 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Jaya kumar, R., Prabaharan, M., Sudheesh Kumar, P. T., Nair, S. V., & Tamurac, H. crosslinked chitosan/poly(ethylene glycol)/ZnO/Ag nanocomposites. Carbohydrate (2011). Biomaterials based on chitin and chitosan in wound dressing applications. Polymers, 89(1), 11–116. Biotechnology Advances, 29(3), 322–337. Liu, X. F., Guan, Y. L., Yang, D. Z., Li, Z., & Yao, K. (2001). Antibacterial action of chitosan Jayakumar, R., Prabaharan, M., Nair, S. V., Tokura, S., Tamura, H., & Selvamurugan, N. and carboxymethylated chitosan. Journal of Applied Polymer Sceince, 79(7), (2010). Novel carboxymethyl derivatives of chitin and chitosan materials and their 1324–1335. biomedical applications. Progress in Materials Science, 55(7), 675–709. Liu, R., Xu, X., Zhuang, X., & Cheng, B. (2014). Solution blowing of chitosan/PVA hy- Jeon, Y. J., Park, P. J., & Kim, S. K. (2001). Antimicrobial effect of chitooligosaccharides drogel nanofiber mats. Carbohydrate Polymers, 101, 1116–1121. produced by bioreactor. Carbohydrate Polymer, 44,71–76. Long, D. D., & Luyen, D. V. (1996). Chitosan-carboxymethylcellulose hydrogels as sup- Jiang, Q., Zhou, W., Wang, J., Tanga, R., Zhang, D., & Wang, X. (2016). Hypromellose ports for cell immobilization. Journal of Macromolecular Science, Part A, 33(12), succinate-crosslinked chitosan hydrogel films for potential wound dressing. 1875–1884. International Journal of Biological Macromolecules, 91,85–91. Lu, G., Ling, K., Zhao, P., Xu, Z., Deng, C., & Zheng, H. (2010). A novel in situ-formed Jothi, N., & Nachiyar, R. K. (2013). Identification and isolation of chitin and chitosan hydrogel wound dressing by the photocross-linking of a chitosan derivative. Wound from cuttle bone of sepia prashadi winckworth, 1936. Global Journal of Biotechnology Repair and Regeneration, 18,70–79. & Biochemistry, 8(2), 33–39. Luo, Y., Kirker, K. R., & Prestwich, G. D. (2000). Cross-linked hyaluronic acid hydrogel Pal, K., Banthia, A. K., & Majumdar, D. K. (2006). Starch based hydrogel with potential. films: New biomaterials for drug delivery. Journal of Controlled Release, 69(1), Biomedical Application as Artificial Skin, 9,23–29. 169–184. Kashyap, N. K. M. R. K. N. (2005). Hydrogels for pharmaceutical and biomedical appli- Ma, Y., Xin, L., Tan, H., Fan, M., Li, J., Jia, Y., et al. (2017). Chitosan membrane dressings cations. Critical Reviews™ in Therapeutic Drug Carrier Systems, 22(2), 107–149. toughened by glycerol to load antibacterial drugs for wound healing. Materials Science Kean, T., & Thanou, M. (2010). Biodegradation, biodistribution and toxicity of chitosan. and Engineering: C, 81, 522–531. Advanced Drug Delivery Reviews, 62,3–11. Macaya, D., & Spector, M. (2001). Injectable hydrogel materials for spinal cord re- Kim, S., & Rajapakse, N. (2005). Enzymatic production and biological activities of chit- generation: A review. Biomedical Materials, 7,1–22. osan oligosaccharides (COS): A review. Carbohydrate Polymers, 62(4), 357–368. MacDonald, S. L., & Pepper, D. S. (1994). Hemoglobin polymerization. Methods in Kim, J. O., Park, J. K., Kim, J. H., Jin, S. G., Yong, C. S., Li, D. X., et al. (2008). Enzymology, 231, 287–308. Development of polyvinyl alcohol–sodium alginate gel-matrix-based wound dressing Maneerung, T., Tokura, S., & Rujiravanit, R. (2008). Impregnation of silver nanoparticles system containing nitrofurazone. International Journal of Pharmaceutics, 359(1-2), into bacterial cellulose for antimicrobial wound dressing. Carbohydrate Polymers, 79–86. 72(1), 43–51. Kim, T. H., An, D. B., Oh, S., Kang, M. K., Song, H. H., & HoLee, J. (2015). Creating Mani, H., Sidhu, G. S., Kumari, R., Gaddipati, J. P., Seth, P., & Maheshwari, R. K. (2002). stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing–thawing Curcumin differentially regulates TGF-β1, its receptors and nitric oxide synthase method to investigate stem cell differentiation behaviors. Biomaterials, 40,51–60. during impaired wound healing. Biofactors, 16,29–43. Kirsner, R. S., & Eaglstein, W. H. (1993). The wound healing process. Dermatologic Clinics, Mason, S., & Clarke, C. (2015). A multicentred cohort evaluation of a chitosan gelling 11(4), 629–640. fibre dressing. British Journal of Nursing, 24(17), 869–876. Kitano, S. S. O. Y. P. R. K. I. Y. O. K. S. Y. O. A. (2006). Genipin suppression of fibrogenic Mazeau, K., Perez, S., & Rainaudo, M. (2000). Predicted influence of N-acetyl group behaviors of the a-TN4 lens epithelial cell line. Journal of Cataract and Refractive content on the conformational extension of chitin and chitosan chains. Journal of Surgery, 32(10), 1727–1735. Carbohydrate Chemistry, 19(9), 1269–1284. Kittur, F. S., Vishu Kumar, A. B., & Tharanathan, R. N. (2003). Low molecular weight McConnell, E. L., Murdan, S., & Basit, A. W. (2008). An investigation into the digestion of chitosans preparation by depolymerization with aspergillus niger pectinase, and chitosan (noncrosslinked and crosslinked) by human colonic bacteria. Journal of characterization. Carbohydrate Research, 338, 1283–1290. Pharmaceutical Sciences, 97, 3820–3829. Kittur, F. S., Vishu Kumar, A. B., Varadaraj, M. C., & Tharanathan, R. N. (2005). Meng, X., Tian, F., Yang, J., He, C. N., Xing, N., & Li, F. (2010). Chitosan and alginate Chitooligosaccharides—Preparation with the aid of pectinase isozyme from asper- polyelectrolyte complex membranes and their properties for wound dressing appli- gillus niger and their antibacterial activity. Carbohydrate Research, 340, 1239–1245. cation. J. Mater. Sci, 21, 1751–1759. Kokabi, M., Sirousazar, M., & Hassan, Z. M. (2007). PVA–clay nanocomposite hydrogels Méthot, S., Changoor, A., Tran-Khanh, N., Hoemann, C. D., Stanish, W. D., Restrepo, A., for wound dressing. European Polymer Journal, 43(3), 773–781. et al. (2016). Osteochondral biopsy analysis demonstrates that BST-CarGel treatment Kong, M., Chen, X. G., Liu, C. S., Yu, L. J., Ji, Q. X., Xue, Y. P., et al. (2008b). Preparation improves structural and cellular characteristics of cartilage repair tissue compared and antibacterial activity of chitosan microspheres in a solid dispersing. Frontiers of with microfracture. Cartilage, 7(1), 16–28. Materials Science in China, 2, 214–220. Mi, F. L., Sung, H. W., & Shyu, S. S. (2000). Synthesis and characterization of a novel Kong, M., Chen, X. G., Xing, K., & Park, H. J. (2010). Antimicrobial properties of chitosan chitosan-based network prepared using naturally occurring crosslinker. Polymer and mode of action: A state of the art review. International Journal of Food Chemistry, 38(15), 2804–2814. Microbiology, 144(1), 51–63. Mi, F. L., Shyu, S. S., & Peng, C. K. (2005). Characterization of ring-opening poly- Kooa, Y. S. H. K. Y. L. S. H. S. K. B. K. C. J. C. L. E. P. H. (2004). Antiinflammatory effects merization of genipin and pH-dependent cross-linking reactions between chitosan of genipin, an active principle of gardenia. European Journal of Pharmacology, 495, and genipin. Polymer Chemistry, 43(10), 1985–2000. 201–208. Milosavljević, N. B., Kljajević, L. M., Popović, I. G., Filipović, J. M., & Kalagasidis Krušić, Koosehgol, S., Ebrahimian-Hossein abadi, M., Alizadeh, M., & Zamanian, A. (2017). M. T. (2010). Chitosan, itaconic acid and poly(vinyl alcohol) hybrid polymer net- Preparation and characterization of in situ chitosan/polyethylene glycol fumarate/ works of high degree of swelling and good mechanical strength. Polymer International, thymol hydrogel as an effective wound dressing. Materials Science and Engineering: C, 59, 686–694. 79,66–75. Mishra, S. K., Mary, D. S., & Kannan, S. (2017). Copper incorporated microporous chit- Koueta, H. V. E. L. B. N. (2014). Applications, uses and By-products from cephalopods. osan-polyethylene glycol hydrogels loaded with naproxen for effective drug release Cephalopod culture. Springer131–147. and anti-infection wound dressing. International Journal of Biological Macromolecules, Krajewska, B. (2001). Diffusional properties of chitosan hydrogel membranes. Journal of 95, 928–937. Chemical Technology and Biotechnology, 76, 636–642. Mo, X., Cen, J., Gibson, E., Wang, R., & Percival, S. L. (2015). An open multicenter Krishna Rao, K. S. V., Naidu, B. V. K., Subha, M. C. S., Sairam, M., & Aminabhavi, T. M. comparative randomized clinical study on chitosan. Wound Repair and Regeneration, (2006). Novel chitosan-based pH-sensitive interpenetrating network microgels for the 23, 518–524. controlled release of cefadroxil. Carbohydrate Polymers, 66, 333–344. Monteiro, O. A. C., & Airoldi, C. (1999). Some studies of crosslinking chitosan-glutar- Kubota, N., Tatsumoto, N., Sano, T., & Toya, K. (2000). A simple preparation of half N- aldehyde interaction in a homogeneous system. International Journal of Biological acetylated chitosan highly soluble in water and aqueous organic solvents. Macromolecule, 26, 119–128. Carbohydrate Research, 324(no. 4), 268–274. Morelli, A., & Chiellini, F. (2010). Ulvan as a New type of biomaterial from renewable Kumbar, S. G., Soppimath, K. S., & Aminabhavi, T. M. (2003). Synthesis and character- resources: Functionalization and hydrogel preparation. Mocromolecular Chemistry and ization of polyacrylamide‐grafted chitosan hydrogel microspheres for the controlled Physics, 211(7), 821–832. release of indomethacin. Journal of Applied Polyner Science, 87, 1525–1536. Morgado, P. I., Miguel, S. P., Correia, I. J., & Aguiar-Ricardo, A. (2017). Ibuprofen loaded Kurita, K. (1998). Chemistry and application of chitin and chitosan. Polymer Degradation PVA/chitosan membranes: A highly efficient strategy towards an improved skin and Stability, 59, 117–120. wound healing. Carbohydrate Polymers, 159, 136–145. Kurita, K., Tomita, K., Tada, T., Ishii, S., Nishimura, S., & shimoda, K. (1993). Squid chitin Morones, J. R., Elechiguerra, J. L., Camacho, A., & Ramirez, J. T. (2005). The bactericidal as a potential alternative chitin source: Deacetylation behavior and characteristic effect of silver nanoparticles. Nanotechnology, 16, 2345–2353. properties. Journal of Polymer Science: Part A Polymer Chemistry, 31(2), 485–491. Moura, M. J., Figueiredo, M. M., & Gil, M. H. (2007). Rheological study of genipin Cross- Kurita, K., Kamiya, M., & Nishimura, S. I. (1991). Solubilization of a rigid polysaccharide: linked chitosan hydrogels. Biomacromolecules, 8, 3823–3829. Controlled partial n-acetylation of chitosan to develop solubility. Carbohydrate Murakami, K., Aoki, H., Nakamura, S., Nakamura, S., Takikawa, M., Hanzawa, M., et al. Polymers, 16,83–92. (2010). Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-im- Li, H., Yang, J., Hu, X., Liang, J., Fan, Y., & Zhang, X. (2011). Superabsorbent poly- paired wound dressings. Biomaterials, 31,83–90. saccharide hydrogels based on pullulan derivate as antibacterial release wound Muzzarelli, R. A. A. (2009a). Genipin-crosslinked chitosan hydrogels as biomedical and dressing. Journal of Biomedical Materials Research, 98A(1), 31–39. pharmaceutical aids. Carbohydrate Polymers, 77(1), 1–9. Lih, E., Seok, J., Kyung, L., Park, M., & Park, K. D. (2012). Rapidly curable chitosan–PEG Muzzarelli, R. A. A. (2009b). Genipin-crosslinked chitosan hydrogels as biomedical and hydrogels as tissue adhesives for hemostasis and wound healing. Acta Biomaterialia, pharmaceutical aids. Carbohydrate Polymers, 77(1), 1–9. 8(9), 3261–3269. Muzzarelli, R. A. A., Mehtedi, M. E., Bottegoni, C., Aquili, A., & Gigante, A. (2015). Liu, Y., & Kim, H. (2012a). Characterization and antibacterial properties of genipin- Genipin-crosslinked chitosan gels and scaffolds for tissue engineering and regenera- crosslinked chitosan/poly(ethylene glycol)/ZnO/Ag nanocomposites. Carbohydrate tion of cartilage and bone. Marine Drugs, 13(no. 12), 7314–7338. Polymers, 89(1), 111–116. MuzzareUi, R. A. A. (1993). Biochemical significance of exogenous chitins and chitosans Liu, Y., & Kim, H. (2012b). Characterization and antibacterial properties of genipin- in animals and patients. Carbohydrate Polymers, 20,7–16.

458 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Nimal, T. R., Baranwal, G., Bavya, M. C., Biswas, R., & Jayakumar, R. (2016). Anti-sta- dried chitosan–polyvinyl pyrrolidone hydrogels as controlled release system for an- phylococcal activity of injectable nano Tigecycline/Chitosan-PRP composite hydrogel tibiotic delivery. Journal of Controlled Release, 68(1), 23–30. using drosophila melanogaster model for infectious wounds. Applied Material & Rocasalbas, G., Francesko, A., Touriño, S., Fernández-Francos, X., Guebitz, G. M., & Interfaces, 8, 22074–22083. Tzanov, T. (2013). Laccase-assisted formation of bioactive chitosan/gelatin hydrogel No, H. K., & Meyers, S. P. (1995). Preparation and characterization of chitin and stabilized with plant polyphenols. Carbohydrate Polymers, 92(no. 2), 989–996. chitosan—A review. Journal of Aquatic Food Product Technology, 4(2). Rosiak, J., Burozak, K., & Pȩkala, W. (1983). Polyacrylamide hydrogels as sustained re- North American Rescue (2018). North American rescue [online]. Available:https://www. lease drug delivery dressing materials. Radition Physics and Chemistry, 22(3-5), narescue.com/hemostatic-bandage-chitogauze-4-x-4yd. 907–915. Nwe, N., Chandrkrachang, S., Stevens, W. F., Maw, T., Tan, T. K., Khor, E., et al. (2002). Ruel-Gariépya, E., Leclair, G., Hildgen, P., Gupta, A., & Leroux, J. C. (2002). Production of fungal chitosan by solid state and submerged fermentation. Thermosensitive chitosan-based hydrogel containing liposomes for the delivery of Carbohydrate Polymers, 49(2), 235–237. hydrophilic molecules. Journal of Controlled Release, 82(2-3), 373–383. Obara, K., Ishihara, M., Ishizuka, T., Fujita, M., Ozeki, Y., Maehara, T., et al. (2003). sam medical (2018). Sam medical. [Online]. Available:https://www.sammedical.com/ Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimu- products/chitosam. lates wound healing in healing-impaired db/db mice. Biomaterials, 24(20), Sannino, A., Demitri, C., & Madaghiele, M. (2009). Biodegradable cellulose-based hy- 3437–3444. drogels: Design and applications. Materials, 2(2), 353–373. Onishi, H., & Machida, Y. (1999). Biodegradation and distribution of water-soluble Schatz, C., Viton, C., Delair, T., Pichot, C., & Domard, A. (2003). Typical physicochemical chitosan in mice. Biomaterials, 20, 175–182. behaviors of chitosan in aqueous solution. Biomacromolecules, 3, 641–648. Patel, S., Srivastava, S., Singh, M. R., & Singh, D. (2017). Preparation and optimization of Segura, T., Anderson, B. C., Chung, P. H., Webber, R. E., Shull, K. R., & Shea, L. D. (2005). chitosan-gelatin films for sustained delivery of lupeol for wound healing. International Crosslinked hyaluronic acid hydrogels: A strategy to functionalize and pattern. Journal of Biological Macromolecules vol. In Press (Available Online). Biomaterials, 26(4), 359–371. Patrulea, V., Ostafe, V., Borchard, G., & Jordan, O. (2015). Chitosan as a starting material Shanmugam, A., Kathiresan, K., & Nayak, L. (2016). Preparation, characterization and for wound healing applications. European Journal of Pharmaceutics and antibacterial activity of chitosan and phosphorylated chitosan from cuttlebone of Biopharmaceutics, 97, 417–426. sepia kobiensis (hoyle, 1885). Biotechnology Reports, 9,25–30. Paul, W., & Sharma, C. P. (2004). Chitosan and alginate wound dressings: A short review. Shibata, H., Heo, Y. J., Okitsu, T., Matsunaga, Y., Kawanishia, T., & Takeuchi, S. (2010). Trends in Biomaterials and Artificial Organs, 18,18–23. Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose Perchyonok, V. T., Reher, V., Zhang, S., Basson, N., & Grobler, S. (2014). Evaluation of monitoring. Proceedings of the National Academy of Science of the United State of Nystatin Containing Chitosan Hydrogels as Potential Dual Action Bio-Active America, 107(42), 17894–17898. Restorative Materials: in Vitro Approach. Journal Og Functional Biomaterials, 5(4), Shukla, R., Kashaw, S. K., Jain, A. P., & Lodhi, S. (2016). Fabrication of apigenin loaded 259–272. gellan gum–chitosan hydrogels (GGCH-HGs) for effective diabetic wound healing. Pillai, C. K. S., Paul, W., & Sharma, C. P. (2009). Chitin and chitosan polymers: Chemistry, International Journal of Biological Macromolecules, 91, 1110–1119. solubility and fiber formation. Progress in Polymer Science, 34(7), 641–678. Silva, L. P., Santos, T. C., Rodrigues, D. B., Pirraco, R. P., Cerqueira, M. T., Reis, R., et al. Pochanavanich, P., & Suntornsuk, W. (2002). Fungal chitosan production and its char- (2017). Stem cell-containing hyaluronic acid-based spongy hydrogels for integrated acterization. Letters in Applied Microbiology, 35(1), 17–21. diabetic wound healing. Journal of Investigative Dermatology, 137(no. 7), 1541–1551. Pulat, M., Kahraman, A. S., Tan, N., & Gümüşderelioğlu, M. (2013). Sequential antibiotic Singh, A., Narvi, S. S., Dutta, P. K., & Pandey, N. D. (2006). External stimuli response on a and growth factor releasing chitosan-PAAm semi-IPN hydrogel as a novel wound novel chitosan hydrogel crosslinked with formaldehyde. Bulletin of Materials Science, dressing. Journal of Biomaterials Science, Polymer Edition, 24(7), 807–809. 29(3), 233–238. Qin, C., Li, H., Xiao, Q., Liu, Y., Zhu, J., & Du, Y. (2006). Water-solubility of chitosan and Smith, T. J., Kennedy, J. E., & Higginbotham, C. L. (2009). The rheological and thermal its antimicrobial activity. Carbohydrate Polymers, 63, 367–374. characteristics of freeze-thawed hydrogels containing hydrogen peroxide for poten- Qiu, Y., & Park, K. (2001). Environment-sensitive hydrogels for drug delivery. Advanced tial wound healing applications. Journal of the Mechanical Behavior of Biomedical Drug Delivery Reviews, 53(3), 321–339. Materials, 2, 264–271. Qu, X., Wirsén, A., & Albertsson, A. C. (2000). Novel pH-sensitive chitosan hydrogels: Souza, R. D., Zahedi, P., Allen, C. J., & Miller, M. P. (2009). Biocompatibility of injectable Swelling behavior and states of water. Polymer, 42, 4589–4598. chitosan-phospholipid implant systems. Biomaterial, 30(23-24), 3818–3824. Qu, X., Wirsén, A., & Albertsson, A.-C. (1999). Synthesis and characterization of pH- Stasica, P., Ciach, M., Radek, M., & Rosiak, J. M. (2000). Approach to construct hydrogel sensitive hydrogels based on chitosan and D,L-lactic acid. Applied Polymer Science, 74, intervertebral disc implants-experimental and numerical investigations. Engineering 3193–3202. Biomaterial, 3(9) p. 9. Racine, L., Texier, I., & Auzély-Velty, R. (2017). Chitosan‐based hydrogels: Recent design Stone, C. A., Wright, H., Clarke, T., Powell, R., & Devaraj, V. S. (2000). Healing at skin concepts to tailor properties and functions. Polymer International, 66(7), 981–998. graft donor sites dressed with chitosan. British Journal of Plastic Surgery, 53(7), Radhakumary, C., Antonty, M., & Sreenivasan, K. (2011). Drug loaded thermoresponsive 601–606. and cytocompatible chitosan based hydrogel as a potential wound dressing. Su, Z., Ma, H., Wu, Z., Zeng, H., Li, Z., Wang, Y., et al. (2014). Enhancement of skin Carbohydrate Polymers, 83(2), 705–713. wound healing with decellularized scaffolds loaded with hyaluronic acid and epi- Rai, M., Yadav, A., & Gade, A. (2009). Silver nanoparticles as a new generation of anti- dermal growth factor. Materials Science and Engineering: C, 44(1), 440–448. microbials. Biotechnology Advances, 27(1), 76–83. Sudheesh Kumar, P. T., Lakshmanan, V., Anilkumar, T. V., Ramya, C., Reshmi, P., Rao, S. B., & Sharma, C. P. (1997). Use of chitosan as a biomaterial: Studies on its safety Unnikrishnan, A. G., et al. (2012). Flexible and Microporous Chitosan Hydrogel/Nano and. J. Biomed. Mater, 34,21–28. ZnO Composite Bandages for Wound Dressing: In Vitro and In Vivo Evaluation. Razzak, D. D. Z. S. M. T. (2001). Irradiation of polyvinyl alcohol and polyvinyl pyrroli- Applied Materials and Interfaces, 4, 2618–2629. done blended hydrogel for wound dressing. Radiation Physics and Chemistry, 62, Sung, H. W., Liang, I. L., Chen, C. N., Huang, R. N., & Liang, H. F. (2001). Stability of a 107–113. biological tissue fixed with a naturally occurring crosslinking agent (genipin). Journal Refojo, M. F., & Leong, F. L. (1981). Poly(methyl acrylate‐co‐hydroxyethyl acrylate) of Biomedical Material, 55, 538–546. hydrogel implant material of strength and softness. Journal of Biomedical Materials Sung, H. W., Huang, R. N., Huang, L. L. H., & Tsai, C. C. (1999). In vitro evaluation of Research Banners, 15(4), 497–509. cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixa- Rhazi, M., Desbrières, J., Tolaimate, A., Alagui, A., & Vottero, P. (2000). Investigation of tion. Biomaterial Science Polymer, 10,63–78. different natural sources of chitin: Influence of the source and deacetylation process Sung, H. W., Chang, Y., Liang, I. L., Chang, W. H., & Chen, Y. C. (2000). Fixation of on the physicochemical characteristics of chitosan. Polymer International, 49(4), biological tissues with a naturally occurring crosslinking agent: Fixation rate and 337–340. effects of pH, temperature, and initial fixative concentration. Journal of Biomedical Ribeiro, M. P., Espiga, A., Silva, D., Baptista, P., Henriques, J., Ferreira, C., et al. (2009). Research, 52,77–87. Development of a new chitosan hydrogel for wound dressing. Wound Repair and Sung, J. H., Hwang, M., Kim, J., Lee, J. H., Kim, Y., Kim, J. H., et al. (2010). Gel char- Regeneration, 17, 817–824. acterisation and in vivo evaluation of minocycline-loaded wound dressing with en- Ribeiro, M. P., Morgado, P. I., Miguel, S. P., outinho, P. C., & Correia, I. J. (2013). hanced wound healing using polyvinyl alcohol and chitosan. International Journal of Dextran-based hydrogel containing chitosan microparticles loaded with growth fac- Pharmaceutics, 392(1–2), 232–240. tors to be used in wound healing. Materials Science and Engineering: C, 33(5), Supper, S., Anton, N., Seidel, N., Riemenschnitter, M., Schoch, C., & Vandamme, T. 2958–2966. (2013). Rheological study of Chitosan/polyol-phosphate systems: Influence of the Ricciardi, R., Auriemma, F., De Rosa, C., & Laupreˆtre, F. (2004). X-ray diffraction ana- polyol part on the thermo-induced gelation mechanism. Langmuir, 29(32), lysis of poly(vinyl alcohol) hydrogels, obtained by freezing and thawing techniques. 10229–10237. Macromolecules, 37, 1921–1927. Tajdini, F., Aminia, M. A., Nafissi-Varcheh, N., & Faramarzi, M. A. (2010). Production, Rickett, T. A., Amoozgar, Z., Tuchek, C. A., Park, J., Yeo, Y., & Shi, R. (2011). Rapidly physiochemical and antimicrobial properties of fungal chitosan from rhizomucor photo-Cross-linkable chitosan hydrogel for peripheral neurosurgeries. miehei and mucor racemosus. International Journal of Biological Macromolecules, Biomacromolecules, 12,57–65. 47(no. 2), 180–183. Rinaudo, M. (2006a). Chitin and chitosan: Properties and applications. Progress in Polymer Tan, S. C., Tan, T. K., Wong, S. M., & Khor, E. (1996). The chitosan yield of zygomycetes Science, 31(7), 603–632. at their optimum harvesting time. Carbohydrate Polymers, 30, 239–242. Rinaudo, M. (2006b). Chitin and chitosan: Properties and applications. Progress in Polymer Tapola, N. S., Lyyra, M. L., Kolehmainen, R. M., Sarkkinen, E. S., & Schauss, A. G. (2008). Science, 31(7), 603–632. Safety aspects and cholesterol-lowering efficacy of chitosan tablets. The Journal of the Rinaudo, M., Pavlov, G., & Desbrie`res, J. (1990). Influence of acetic acid concentration American College of Nutrition, 27,22–30. on the solubilization of chitosan. Polymer, 40, 7029–7032. Tayel, A. A., Moussa, S., Opwis, K., Knittel, D., Schollmeyer, E., & Nickisch-Hartfiel, A. Risbud, M. K., & Bhonde, R. R. (2000). Polyacrylamide-chitosan hydrogels: In vitro bio- (2010). Inhibition of microbial pathogens by fungal chitosan. International Journal of compatibilityand sustained antibiotic release studies. Drug Delivery, 7,69–75. Biological Macromolecules, 47(1), 10–14. Risbud, M. V., Hardikar, A. A., Bhat, S. V., & Bhonde, R. R. (2000). pH-sensitive freeze- Tiena, C., Lacroix, M., Ispas-Szabo, P., & Mateescu, M. A. (2003). N-acylated chitosan:

459 H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Hydrophobic matrices for controlled drug release. Journal of Controlled Release, 93(1), healing properties of PVA/ws-chitosan/glycerol hydrogels made by irradiation fol- 1–13. lowed by freeze–thawing. Radiation Physics and Chemistry, 79(5), 606–611. Tomihata, K., & Ikada, Y. (1997). In vitro and in vivo degradation of films of chitin and its Yang, D. H., Seo, D. I., Lee, D., Bhang, S. H., Park, K., Jang, G., et al. (2017). Preparation deacetylated derivatives. Biomaterials, 18, 567–573. and evaluation of visible-light cured glycol chitosan hydrogel dressing containing Tsai, M. L., Bai, S. W., & Chen, R. H. (2008). Cavitation effects versus stretch effects dual growth factors for accelerated wound healing. Journal of Industrial and resulted in different size and polydispersity of ionotropic gelation chitosan–sodium Engineering Chemistry, 53, 360–370. tripolyphosphate nanoparticle. Carbohydrate Polymers, 71, 448–457. Yang, W., Fortunati, E., Bertoglio, F., Owczarek, J. S., Bruni, G., Kozanecki, M., et al. Tsai, T. H., Westly, J., Lee, T. F., & Chen, C. F. (1994). Identification and determination of (2018). Polyvinyl alcohol/chitosan hydrogels with enhanced antioxidant and anti- geniposide, genipin, gardenoside, and geniposidic acid from herbs by HPLC/photo- bacterial properties induced by lignin nanoparticles. Carbohydrate Polymers, 181, diode-array detection. Journal of Liquid Chromatography, 17, 2199–2205. 275–284. Tsao, C. T., Chang, C. H., Lin, Y. Y., Wu, M. F., Wang, J., Han, J. L., et al. (2010). Yaoa, C., Liua, B., & Hsuc, S. (2005). Calvarial bone response to a tricalcium phosphate- Antibacterial activity and biocompatibility of a chitosan–gamma-poly(glutamic acid) genipin crosslinkedgelatin composite. Biomaterials, 3065–3074 p. 26. polyelectrolyte complex hydrogel. Carbohydrate Research, 345, 1774–1780. Yen, M. T., & Mau, J. L. (2007). Physico-chemical characterization of fungal chitosan Tyliszczak, B., Drabczyk, A., Kudłacik-Kramarczyk, S., Bialik-W˛as, K., Kijkowska, R., & from shiitake stipes. LWT – Food Science and Technology, 40, 472–479. Sobczak-Kupiec, A. (2017). Preparation and cytotoxicity of chitosan-based hydrogels Yenilmez, E., Basaran, E., Arslan, R., Berkman, M. S., Güven, U. M., Baycu, C., et al. modified with silver nanoparticles. Colloids and Surfaces B: Biointerfaces, 160(1), (2015). Chitosan gel formulations containing egg yolk oil and epidermal growth 325–330. factor for dermal burn treatment. Pharmazie, 70,67–73. Ueno, H., Mori, T., & Fujinaga, T. (2001). Topical formulations and wound healing ap- Yu, L., & Ding, J. (2008). Injectable hydrogels as unique biomedical materials. Chemical plications of chitosan. Advanced Drug Delivery Reviews, 52(2), 105–115. Society Reviews, 37, 1473–1481. Ueno, K., Yamaguchi, T., Sakairi, N., Nishi, N., & Tokura, S. (1997). Antimicrobial activity Zargar, V., Asghari, M., & Dashti, A. (2015). A review on chitin and chitosan polymers: by fractionated chitosan oligomers. Advances in Chitin Science, 2, 156–161. Structure, chemistry, solubility, derivatives, and applications. ChemBioEng Reviews, 2, Valentine, R., Athanasiadis, T., Moratti, L., Hanton, L., Robinson, S., & Wormald, P. J. 1–24. (2010). The efficacy of a novel chitosan gel on hemostasis and wound healing after Zhai, M., Yoshii, F., Kume, T., & Hashim, K. (2002). Syntheses of PVA/starch grafted endoscopic sinus surgery. American Journal of Rhinology & Allergy, 24(1), 70–75. hydrogels by irradiation. Carbohydrate Polymers, 50(3), 295–303. Vårum, K. M., Myhr, M. M., Hjerde, R. J. N., & Smidsrød, O. (1997). In vitro degradation Zhang, H., & Neau, S. H. (2011). In vitro degradation of chitosan by a commercial enzyme rates of partially N-acetylated chitosans in human serum. Carbohydrate Research, preparation: Effect of molecular weight and degree of deacetylation. Biomaterials, 299(1-2), 99–101. 22(12), 1653–1658. Varum, K. M., Ottoy, M. H., & Smidsrod, O. (1994). Water-solubility of partially N- Zhang, Y., Thomas, Y., Kim, E., & Payne, G. F. (2012). pH- and voltage-responsive chit- acetylated chitosans as a function of pH: E ffect of chemical composition and depo- osan hydrogel through covalent Cross-linking with catechol. Journal of Physical lymerisation. Carbohydrate Polymer Journal, 25,65–70. Chemistry B, 116, 1579–1585. Vasile, B. S., Oprea, O., Voicu, G., Ficai, A., Andronescu, E., Teodorescu, A., et al. (2014). Zhang, Y., Guan, Y., & Zhou, S. (2005). Single component chitosan hydrogel microcapsule Synthesis and characterization of a novel controlled release zinc oxide/gentami- from a layer-by-layer approach. Biomacromolecules, 6, 2365–2369. cin–chitosan composite with potential applications in wounds care. International Zhang, C. Y., Parton, L. E., Ye, C. P., Krauss, S., Shen, R., Lin, C. T., et al. (2006). Genipin Journal of Pharmaceutics, 463(2), 161–169. inhibits UCP2-mediated proton leak and acutely reverses obesity- and high glucose- Velnar, T., Bailey, T., & Smrklj, V. (2009). The wound healing process: An overview of the induced beta cell dysfunction in isolated pancreatic islets. Cell Metabolism, 3, cellular and molecular mechanisms. The Journal of International Medical Research, 37, 417–427. 1528–1542. Zhang, H., Zhang, F., & Wu, J. (2013). Physically crosslinked hydrogels from poly- Verheul, R. J., Amidi, M., van Steenbergen, M. J., van Riet, E., Jiskoot, W., & Hennink, W. saccharides prepared by freeze–thaw technique. Reactive and Functional Polymers, 73, E. (2009). Influence of the degree of acetylation on the enzymatic degradation and in 923–928. vitro biological properties of trimethylated chitosans. Biomaterials, 30, 3129–3135. Zhang, D., Zhou, W., Wei, B., Wang, X., Tanga, R., Nie, J., et al. (2015). Carboxyl-mod- Vimala, K., Mohan, Y. M., Varaprasad, K., Redd, N. N., Ravindra, S., Naidu, N. S., et al. ified poly(vinyl alcohol)-crosslinked chitosan hydrogel films for potential wound (2011). Fabrication of curcumin encapsulated chitosan-PVA silver nanocomposite dressing. Carbohydrate Polymers, 125, 189–199. films for improved antimicrobial activity. Journal of Biomaterials and Zhang, L., Ma, Y., Pan, X., Chen, S., Zhuang, H., & Wang, S. (2018). A composite hydrogel Nanobiotechnology, 2,55–64. of chitosan/heparin/poly (γ-glutamic acid) loaded with superoxide dismutase for Wahid, F., Wang, H., Lu, Y., Zhong, C., & Chu, L. (2017). Preparation, characterization wound healing. Carbohydrate Polymers, 180, 168–174. and antibacterial applications of carboxymethyl chitosan/CuO nanocomposite hy- Zhao, H. M. M. Z. F. Y. N. N. T. K. L. (2003). Synthesis of antibacterial PVA/CM-chitosan drogels. International Journal of Biological Macromolecules, 101, 690–695. blend hydrogels with electron beam irradiation. Carbohydrate Polymers, 53(4), Wang, T., Zhu, X., Xue, X., & Wu, D. (2012). Hydrogel sheets of chitosan, honey and 439–446. gelatin as burn wound dressings. Carbohydrate Polymers, 88(1), 75–83. Zhao, L., & Mitomo, H. (2008). Synthesis of pH-sensitive and biodegradable CM- White, S. A., Farina, P. R., & Fulton, I. (1979). Production and isolation of chitosan from Cellulose/Chitosan polyampholytic hydrogels with electron beam irradiation. Journal mucor rouxii. Applied and Environmental Microbiology, 38, 323–328. of Bioactive and Compatible Polymers, 23, 319–333. Wong, V. W., Rustad, K. C., Glotzbach, J. P., Sorkin, M., Inayathullah, M., Major, M. R., Zhou, H. Y., Chen, X. G., Kong, M., Liu, C. S., Cha, D. S., & Kennedy, J. F. (2008). Effect of et al. (2011). Pullulan hydrogels improve mesenchymal stem cell delivery into High- molecular weight and degree of chitosan deacetylation on the preparation and oxidative-stress wounds. Micromolecular Bioscience, 11, 1458–1466. characteristics of chitosan thermosensitive hydrogel as a delivery system. Xiaomin, Y., Qi, L., Xiliang, C., Feng, Y., & Zhiyong, Z. (2008). Investigation of PVA/ws- Carbohydrate Polymers, 73, 263–273. chitosan hydrogels prepared by combined y-irradiation and freeze-thawing. Zhou, Y., Zhao, Y., Wang, L., Xu, L., Zhai, M., & Wei, S. (2012). Radiation synthesis and Carbohydrate Polymers, 73, 401–408. characterization of nanosilver/gelatin/carboxymethyl chitosan hydrogel. Radiation Xie, W. M., Xu, P. X., Wang, W., & Lu, Q. (2001). Antioxidant activity of water-soluble Physics and Chemistry, 81(5), 553–560. chitosan derivatives. Bioorganic & Medicinal Chemistry Letters, 11, 1699–1703. Zou, X., Zhao, X., Ye, L., Wang, Q., & Li, H. (2015). Preparation and drug release behavior Xinyin, L. (2003). Drug delivery systems based on polymer blends: Synthesis, characterization of pH-responsive bovine serum albumin-loaded chitosan microspheres. Journal of and application, a thesis. Drexel University. Industrial and Engineering Chemistry, 21, 1389–1397. Y.B, S., Gurny, R., & Jordan, O. (2008). A novel thermoresponsive hydrogel based on D. Y. S. J. C. M. E. H. B. H. Thomas, HYDROGEL IMPLANT. US Patent US 2006/0224244 chitosan. European Journal of Pharmaceutics and Biopharmaceutics, 68(1), 19–25. A1, 5 Oct 2006. Yang, X., Yang, K., Wu, S., Chen, X., Yu, F., Li, J., et al. (2010). Cytotoxicity and wound

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