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Carbohydrate Polymers

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Chitosan based hydrogels and their applications for drug delivery in wounddressings: A review

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

A R T I C L E I N F O

Keywords:Chitosan hydrogelWound dressingDrug deliveryGrowth factorNanoparticles

A B S T R A C T

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

1. Introduction

Hydrogels are three-dimensional, cross-linked networks which canabsorb and retain significant amounts of water, without dissolving orlosing their three dimensional structures (Ahmed, 2015; Kashyap, 2005;Y.B. et al., 2008). The gelation and biodegradation are two key factorsaffecting the fate of cells (Li et al., 2012). Moreover, some hydrogelshave antibacterial and antifungal activities (Jaya kumar et al., 2011),and these properties could be useful for wound dressings and accel-erating the wound healing process (Ueno et al., 2001). Polymers thatform hydrogels may have hydrophilic or hydrophobic functionalgroups. Hydrophilic functional groups, such as hydroxyl (−OH), amine(NH2) and amide (−CONH−CONH2) enable the hydrogel to absorbwater leading to hydrogel expansion, which is known as swelling.During swelling, the cross-linked structure of hydrogels prevents com-plete destruction of the hydrogel cross-links and dissolution. Hydrogelswith hydrophobic chains such as poly lactic acid have lower waterswelling capacity than hydrophilic lattices. Hydrogels can be preparedfrom synthetic or natural polymers, involving a wide range of chemicalcompositions and with different mechanical, physical and chemicalproperties. Natural polymers such as alginate (Balakrishnan et al.,2005; Kamoun et al., 2015; Kim et al., 2008; Murakami et al., 2010),chitosan (Milosavljević et al., 2010; Racine et al., 2017), hyaluronicacid (Luo et al., 2000; Segura et al., 2005; Silva et al., 2017) cellulose(Abd El-Mohdy, 2013; Chang et al., 2009; Maneerung et al., 2008;Sannino et al., 2009), starch (Pal et al., 2006; Pal et al., 2006; Zhaiet al., 2002), ulvan (Alvesab et al., 2012; Morelli & Chiellini, 2010),gelatin (Draye, Delaey, Voorde, Bulcke, Reu et al., 1998; Wang et al.,

2012), pullulan (Li et al., 2011; Wong et al., 2011) and/or syntheticpolymers like polyvinyl alcohol (Kokabi et al., 2007; Razzak, 2001;Yang et al., 2008), polyacrylamide (Ezra et al., 2009; Risbud & Bhonde,2000; Rosiak et al., 1983) and polyethylene glycol (Ajji et al., 2005;Gupta et al., 2011; Lih et al., 2012) form hydrogels.

Hydrogels are classified into two categories: chemical or permanentgels that have covalently bonded cross-linked networks (replacing hy-drogen bonds by strong and stable covalent bonds) and physical orreversible gels whose networks are held together by molecular en-tanglements, and/or secondary forces including ionic, hydrogenbonding or hydrophobic interactions (Ahmed, 2015). In physicallycross-linked gels, dissolution is prevented by physical interactions,which exist between different polymer chains (Hennink & Nostrum,2002). Hydrogels can be formulated in a variety of physical shapes,including slabs, microparticles, nanoparticles, coatings, and films(Hoare & Kohane, 2008).

The most important property of hydrogels is their biocompatibility,which can be defined as the ability of a material to be in contact withbodily organs with minimum damage to the surrounding tissues andwithout triggering undesirable immune responses (Caló &Khutoryanskiy, 2015). In some cases this property has been taken ad-vantage of in wound dressings, because after healing it prevents ex-cessive tissue granulation and scar formation (Chung et al., 1994),though they can be hard to handle, may be difficult to load with drugsand sterilize, and usually have low mechanical strength (Huffman,2012). In spite of these disadvantages, hydrogels have many applica-tions in different fields including tissue engineering, pharmaceuticalsand biomedical engineering, in the forms of wound dressings (Benamer

https://doi.org/10.1016/j.carbpol.2018.06.114Received 19 March 2018; Received in revised form 25 June 2018; Accepted 26 June 2018

⁎ Corresponding author.E-mail address: atonelli@ncsu.edu (A.E. Tonelli).

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et al., 2006; Kokabi et al., 2007; Lu et al., 2010), drug delivery vehicles(Hoare & Kohane, 2008; Qiu & Park, 2001; Xinyin, 2003), implants(Refojo & Leong, 1981; Stasica et al., 2000; ZZZZZ, 2018), and in-jectable polymeric systems (J.P. & T.H., 2006; Macaya & Spector, 2001;Shibata et al., 2010; Yu & Ding, 2008).

Chitosan is one of the most widely used materials for making hy-drogels, and has been considered for wound dressing applications. Ithas excellent biocompatibility, low toxicity and immune-stimulatoryactivities (Li, Rodrigues et al., 2012; Souza et al., 2009). Due to theseproperties, chitosan shows good biocompatibility and positive effectson wound healing. It can also accelerate repair of different tissues andfacilitate contraction of wounds (Jaya kumar et al., 2011). The Cyto-compatibility of chitosan has also been studied and shown that it doesnot have acute cytotoxic effect (Gonçalves Ferreira et al., 2016). On theother hand, as a natural based compound, chitosan may be con-taminated by organic and inorganic impurities, and presents broadpolydispersity. Chitosan is poorly soluble, except in acidic medium,which makes analyses difficult to perform (Croisier & Jérôme, 2013).

Chitosan also has been used in commercial wound dressings such asChitoSam™ (sam medical, 2018), ChitoGauze XR pro (North AmericanRescue, 2018), ChitoFlex (H.M.T. Inc., 2007), and Axiostat® (AXIO,2018) which are high-performance hemostatic dressings.

This review emphasizes on chitosan hydrogels and their applica-tions in wound dressing and drug delivery. Initially chitosan and itsproperties are described and different kinds of hydrogel preparationmethods are discussed. In the second section the utilization of chitosanhydrogels are summarized. Finally, utilization of some drugs such asgrowth factors, nano particles and chemicals in chitosan hydrogels aredescribed.

2. Chitosan

Due to its reactive amino groups, chitosan is the only natural ca-tionic polymer and has many commercial applications: it accelerateswound healing, is antimicrobial, anticoagulant, antibacterial, anti-fungal, anti-tumor, and haemostatic [62,63]. The generic term chitosandescribes a range of presumably pure poly-(beta-1-4) N-acetyl-D-glu-cosamine materials (shown in Fig. 1) whose properties are highly de-pendent on its degree of deacetylation; average molecular weight,polydispersity, and its structure.

(Zhou et al. (2008)) investigated the effect of molecular weight anddegree of deacetylation on chitosan hydrogels and concluded that thesetwo parameters affected the pH values, turbidity, viscosity, and ther-mosensitive characteristics of the product hydrogels. Degree of deace-tylation affects chitosan antimicrobial activity (Chung et al., 2004; Liuet al., 2001); on the other hand chitosan microspheres with a high

degree of deacetylation (97.5%) lead to higher positive charge density,which confers stronger antibacterial activity than moderate degree ofdeacetylation (83.7%) against Staphylococcus aureus at pH 5.5 (Konget al., 2008b). It was reported that polydispersity is a useful quantitywhich can clarify the degradation mechanism (Chen et al., 1997).Thedecrease of polydispersity as degree of deacetylation increases couldappear as surprising since the deacetylation reaction does not degradechitosan chains (Schatz et al., 2003). Min et al. (Tsai et al., 2008) havestudied factors that are effective on chitosan polydispersity, such as,solution temperature and solution concentration of chitosan. They alsoexplained why the polydispersities of chitosan nanoparticles obtainedby ultrasonic treatment or by mechanical shearing were similar. Thestructures of chitosan hydrogels can affect their swelling behaviors,because swelling behavior reflects the amount of network porosity andmesh size of the network, which may also indicate its drug releasebehavior (Berger et al., 2004). Porosity of chitosan membranes could bemodified by crosslinking with some materials such as glutaraldehyde(Krajewska, 2001).

Chitosan under acidic condition also has a high capacity for en-trapping various metal ions including (Ni2+, Zn2+, Co2+, Fe2+, Mg2+

and Cu2+) (Kurita, 1998). Rainaudo et al. (Mazeau et al., 2000) havestudied chitosan chain stiffness, and concluded that chitin and chitosanare semi-rigid polymers characterized by a persistence length (asymp-totic value obtained at high degree of polymerization) that dependsmoderately on the degree of acetylation of the molecule.

2.1. Sources of chitosan

Chitin, poly (β-(1→4)-N-acetyl-D-glucosamine) is a natural poly-saccharide, first identified in 1811 by French chemist Henri Braconnot(Arias et al., 2004). Chitin forms a part of the extracellular matrix ofcertain living organisms as a proteoglycan. It is the most abundantpolymer after cellulose. This biopolymer is synthesized by an enormousnumber of living organisms among which are insects (cuticles) andcrustaceans (skeletons), e.g., crab, shrimp and lobster. Cephalopod by-products are a valuable source of chitin, polyunsaturated acids, andcollagen. (Koueta, 2014). Chitin is also extracted from the exoskeletonof cephalopod species, such as squid, cuttlefish and octopi (Arrouzeet al., 2017; Jothi & Nachiyar, 2013; Kurita et al., 1993; Shanmugamet al., 2016). (See Fig. 1).

Chitosan, which was discovered and named in 1859 by Roget(Patrulea et al., 2015), is obtained by partial deacetylation of chitinunder alkaline conditions and is the most important chitin derivative interms of applications (Rinaudo, 2006b). (See Fig. 1). The physico-chemical characteristics of the obtained chitosan are closely related tothe taxonomy of the marine sources (Rhazi et al., 2000). Chitosan can

Fig. 1. Chitosan structure.

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also be produced by enzymatic rather than alkali treatment at hightemperatures (Cai et al., 2006; No & Meyers, 1995). Chitosan can beenzymatically degraded by chitinases and chitosanases but these en-zymes are unavailable in bulk quantities for commercial applications. Ithas been reported that β-glucosidase from almond emulsin can hydro-lyze chitin substrates owing to an existing chitinase in the enzymepreparation (Zhang & Neau, 2011). It has been reported that chitinasesare not responsible for the in vitro degradation of chitosans in humanserum (Vårum et al., 1997). Enzymatic degradation minimizes adversechemical modifications of products and promotes their biological ac-tivities. However, higher costs of hydrolytic enzymes limit the appli-cation of enzymatic methods. To reduce this production cost, reuse ofhydrolytic enzymes may be recommended (Kim & Rajapakse, 2005).

Chitosan preparation from fungal cell walls with fermentationtechnology has become an alternative economical way for the pro-duction of this polymer. It can be found in the cell wall of certaingroups of fungi, particularly zygomycetes (Nwe et al., 2002; Tayel et al.,2010). There are several advantages of using these fungi to producechitosan. The most important is that the cell wall of zygomycetous fungicontains a large quantity of chitosan and the physicochemical proper-ties of this chitosan can be manipulated and standardized by controllingthe parameters of fermentation. For example, different molecularweight chitosans can be produced when these fungi are grown on atdifferent pHs and on media with different compositions (Tan et al.,1996). Sufficient amounts of chitosan have been identified in Mucorrouxii (White et al., 1979), phycomyces, and saccharomyces (Arcidiacono& Kaplan, 1992). In another study, chitosans with high degree of dea-cetylation (above 97%) were extracted from R.miehei and M.racemosus(Tajdini et al., 2010).

An investigation was performed to evaluate the chitosan char-acterization produced by several species of fungi. Comparison of fungalchitosan with a commercial one derived from crab shells showed lowerdegree of deacetylation, viscosity and molecular weight of the fungalchitosan. Chitosan with a low molecular weight reduces the tensilestrength and elongation of the chitosan membrane, but increase itspermeability. Fungal chitosan may have potential medical and agri-cultural applications (Pochanavanich & Suntornsuk, 2002). This is ingeneral agreement with the findings of Yen et al. (Yen & Mau, 2007;Yen, Yang, and Mau, (2009)).

2.2. Solubility of chitosan

Due to the high cohesive energy of chitin, which is related to strongintermolecular interactions from hydrogen bonds, chitin is insoluble inmany typical solvents, such as water, dilute acids and alcohols (Zargaret al., 2015). This is a major problem that limits the development ofprocessing and usage of chitin.

Chitosan, the most important derivative of chitin, is soluble in diluteaqueous acidic media at a degree of deacetylation of 50% and higher(depending on the origin of the polymer) due to its primary aminogroups that have a pKa value of 6.3. Solubilization occurs by protona-tion of the –NH2 group of the D-glucosamine repeating unit, wherebythe polysaccharide is converted to a polyelectrolyte in acidic media.Solubility of chitosan is usually examined in acetic acid by dissolving itin 1% or 0.1 M acetic acid (Rinaudo, 2006a). Rinaudo et al.. (Rinaudo,Pavlov, and Desbrie`res, (1990)) demonstrated that the amount of acidneeded depends on the quantity of chitosan to be dissolved. The con-centration of protons needed is at least equal to the concentrationof−NH2 units involved. As the degree of protonation is progressivelyincreased, so is the solubilization of chitosan. The effect of depoly-merization on the solubility of the chitosans with different degree ofdeacetylation at neutral pH-values was investigated by Vårum et al(Varum et al., 1994). They emphasized that the solubility differencesbetween chitosans with different degree of deacetylation may haveprofound influence on accessibility of chitosans to enzymes (many en-zyme assays are performed at pH 6.5) and the biological effects of

chitosans (testing of chitosans in vivo and in vitro are often performed atneutral pH-values).

Sorlier et al. (Sorlier, Viton, and Domard, (2002)) demonstrated thatwhen the degree of dissociation (α) in solution increases, the role of thecationicity of the amine groups, which depends on the degree of acet-ylation, plays a more important role on the behavior of the polymerchains. They showed that molecular weight and degree of deacetylationhave important effect on chitosan solubility.

Solubility of chitosan will be enhanced by decreasing its molecularweight. Molecular weight changes the content of N-acetylglucosaminesunits in chitosan, which will have intramolecular as well as an inter-molecular influences, resulting in different chitosan conformations.However, improving solubility by controlling deacetylation comes atthe cost of low yield (Kurita et al., 1991). Sogias et al.. (Sogias, Khu-toryanskiy, and Williams, (2010)) showed that break down of chitosancrystallinity will expand the range of chitosan solubility. They studiedtwo approaches for improving chitosan solubility, physical and che-mical methods. In their chemical approach, re-acetylation improvedchitosan solubility up to pH=7.4. The physical approach included theuse of additives, such as urea, and guanidine hydrochloride, with theabilities to disrupt intra- and inter macromolecular hydrogen bonding.In the presence of 8mol L−1 urea and 1.5mol L−1 guanidine hydro-chloride the solubility of chitosan shifted to pH=8.0 and 6.5, respec-tively.

Introducing small chemical groups to the chitosan structure, such asalkyl (hydroxypropyl chitosan (Xie et al., 2002)) or carboxymethylgroups can drastically increase the solubility of chitosan (Jayakumaret al., 2010; Pillai et al., 2009). A simple and improved method ofpreparing highly soluble chitosan (half N-acetylated chitosan) was de-veloped using a series of chitosan samples of low molecular weights(Kubota et al., 2000).

2.3. Toxicity

There are various studies on chitosan toxicity. Scientists have donesome studies on chitosan toxicity in guinea pigs (Aspden et al., 1997),frog, human nasal palate tissue (Aspden et al., 1994; Aspden et al.,1995), and the nasal membranes of rats (Aspden et al., 1996). In all ofthe tests, toxicity was negligible. Ribeiro et al. (Ribeiro et al. (2009))studied the in vitro cytotoxicity of chitosan hydrogels tested with dermalfibroblasts obtained from rat skin. Their cell viability study showed thatthe hydrogel and its degradation by-products are non-cytotoxic. His-tological analysis also revealed lack of a reactive or a granulomatousinflammatory reaction in skin lesions with chitosan hydrogels, and theabsence of pathological abnormalities in the organs supported the localand systemic histocompatibility of this biomaterial.

in vivo chronic toxicity studies reported by Carreño-Gómez et al.(Carreño-Gómez & Duncan, 1997) have shown that intravenous ad-ministration of chitosan (4.5 mg/kg/day for 11 days) to rabbits did notlead to any abnormal changes. Data on chitosan toxicity from humanstudies in general are quite limited. Gades et al. (Gades & Stern, 2003)reported that quantities exceeding 4.5 g chitosan taken daily by humanvolunteers did not result in toxic effects. Even higher oral levels of up to6.75 g were reported as safe. It has been reported that short-termhuman trials of up to 12 weeks have shown no clinically significantsymptoms, including no evidence of an allergic response (Tapola et al.,2008). Chitosan mouthwash safety was evaluated through Ames, MTTand V79 chromosomal aberration assays. The chitosan mouthwashpossessed lower toxicity and higher antimicrobial activity than thecommercial mouthwash tested. Furthermore, while it is capable ofcompletely inhibiting the two selected pathogenicity markers (strep-tococci and enterococci), contrary to the commercial mouthwash, it didnot cause major reductions in the viability of the normal oral microflora(Costa et al., 2014).

Ravindranathan et al. (Ravindranathan, Koppolu, Smith, andZaharoff, (2016)) Studied purified, low endotoxin chitosan, and

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determined that viscosity/molecular weight and degree of deacetyla-tion within the ranges of 20–600 CP and 80–97%, respectively, have noimpact on chitosan’s immunoreactivity. Endotoxin contamination wasexpected to be a major factor influencing immunoreactivity. Their re-sults indicated that only endotoxin content and not degree of deace-tylation or viscosity influenced chitosan-induced immune responses.Their data also indicated that low endotoxin chitosan (< 0.01 EU/mg)with viscosities of 20–600 cP and 80%–97% degree of deacetylation areessentially inert. This study highlights the need for more completecharacterization and purification of chitosan in preclinical studies be-fore being used in clinical application.

Baldrick (Baldrick (2010)) implies that chitosan can be used as asafe pharmaceutical excipient for non-parenteral and non-blood con-tact. Based on the available data, safe use of chitosan as a parenteralexcipient is not clear. Its use as a medical device to control bleedingrelies on the haemostatic biological nature of the material and studieshave demonstrated local findings, such as blood coagulation, thrombusformation and platelet adhesion/aggregation. In spite of some in vitrostudies related to cytotoxicity findings (Baldrick (2010)), there are re-ports of non-toxic usages of chitosan in pharmaceuticals, such as to stopbleeding (Dowling et al., 2011; Horio et al., 2010; Valentine et al.,2010).

2.4. Biomedical properties

The degradation rate of chitosan can be controlled by changing itsdegree of deacetylation and/or its molecular weight (Tomihata & Ikada,1997). Many researches have been done to prove the degraded productsof chitosan are nontoxic, non-immunogenic, and non-carcinogenic(MuzzareUi, 1993). Risbud et al. (Risbud & Bhonde, 2000) reportedcharacterization, biocompatibility, and release properties of poly-acrylamide/chitosan hydrogels and showed that chitosan possess ex-cellent biocompatibility and bioabsorbability. In this research, in vitrocytotoxicity studies using a colorimetric assay for assessing cell meta-bolic activity, membrane permeability, and lysosomal activity of cells(MTT and NR assays) showed no toxic effects on different cells in up to40% hydrogel extract.

The antimicrobial properties of chitosan and its derivatives haveattracted great attention from researchers (Kong et al., 2010). Anti-microbial activity of chitosan has been demonstrated against manybacteria, filamentous fungi, and yeasts. Chitosan has a wide spectrum ofactivity and high killing rates against gram-positive and gram-negativebacteria (Ueno et al., 1997), with low toxicity toward mammalian cells.

Antibacterial effects of chitosan are reported to be dependent on itsmolecular weight (Jeon et al., 2001). To prove this idea, No et al. (No,Park, Lee, and Meyers, (2002)) examined the antibacterial activity ofsix chitosans and six chitosan oligomers with widely different molecularweights against gram-negative and gram-positive bacteria. Theyshowed that chitosan markedly inhibited the growth of most bacteriatested, although their inhibitory effects differed with molecular weightand the particular bacterium. Chitosan generally showed strongerbactericidal effects for gram-positive bacteria than for gram-negativebacteria. The reason for this difference is unclear, but Y. J. Jeon et. al(Jeon et al., 2001) showed the oligosaccharides as well as chitosanexhibited better inhibitory effects against Gram-positive compared withGram-negative bacteria. Thus, it might be reasonable that chitosanshowed remarkable inhibitory effect against the growth of lactic acidbacteria which are Gram-positive.

Sizes of chitosan particles play an important role in inflammasomeactivation. The inflammasome is a cytosolic complex which is re-sponsible for the activation of inflammatory responses, such as IL-1β.Glycans passed through filters of defined size, showed that the smallestsize fraction (< 20 μm) induced the most cleavage of pro-IL-1β andrelease of the mature cytokine. The larger-sized fractions also had somebioactivity, which may have been due to some smaller particles thatfailed to pass through the filters. Partial digestion of chitosan with

pepsin, an enzyme that breaks down proteins into smaller peptides,enhanced the ability of the glycan to activate the inflammasome (Bueteret al., 2011).

Biodegradability of chitosan has been studied by many researchers.It was found that lysozyme (Onishi & Machida, 1999), proteases (Rao &Sharma, 1997) and porcine pancreatic enzymes (Verheul et al., 2009)are able to degrade Chitosan under specific conditions. Pectinase iso-zyme from Aspergilus niger has also been shown to digest chitosan atlow pH providing lower Mw chitosans (Kittur et al., 2003; Kittur et al.,2005). Connell et al. (McConnell et al., 2008) used human fecal pre-parations and showed significant degradation of chitosan films, glu-taraldehyde crosslinked films, and tripolyphosphate crosslinked films.Brenner et al. (Brenner, Hostetter, and Humes, (1978)) demonstratedthat many enzymes seem to show an activity against chitosan, at least invitro, but derivatives could give indigestible molecules. To be clinicallyviable, these compound particles should remain small enough (< 42 Åradius for neutral molecules) to be renally excreted. The bio-distribu-tion is controllable through the route of administration, dosage formand chitosan characteristics, i.e., a film/dense matrix will persist at thesite of administration due to the physical characteristics. The de-gradation of the matrix is controllable through crosslinking modifica-tion of the amine groups. Intravenous distribution can be adjusted byparticle size (< 100 nm), and molecular weight can determine thestability of the particle (Kean & Thanou, 2010).

2.5. pH sensitivity

The numerous amine groups on the chitosan backbone confer a pH-dependent solubility, which has been utilized to fabricate biosensorsand drug delivery systems. Chitosan properties can be convenientlyaltered by derivatization of its hydroxyl and amino groups (Zhang et al.,2012). The amino groups of chitosan are ionized in acidic buffers,which contribute to the electrostatic repulsion between adjacent io-nized –NH2 groups and lead to chain expansion and eventually increasethe water absorption of the chitosan gels. The swelling degree of theionic pH-sensitive hydrogel is mainly determined by the degree of io-nization (Guanghua et al., 2008). Some attempts have been made forregulating chitosan pH-sensitivity by adding different additives orgrafting or mixing with other polymers and preparing Polyelectrolytecomplexes with anionic polymers (Krishna Rao et al., 2006; Meng et al.,2010; Qu et al., 1999; Qu et al., 2000). The nature of the pH response ofchitosan and its derivatives has been described by several researchers.Islam et al. (Islam & Yasin, 2012) synthesized a pH sensitive blendbased on chitosan and poly vinyl alcohol (PVA) using a nontoxic crosslinker for a drug delivery system. They concluded that the pH sensi-tivity and biocompatibility properties of this chitosan/PVA hydrogelmade it suitable for medicinal and pharmaceutical applications. Anti-microbial activity of chitosan is also pH dependent, it has been reportedthat chitosan displayed antibacterial activity only in an acid environ-ment (Helander et al., 2001).

3. Chitosan hydrogel preparation methods

3.1. Chemical Cross linking (permanent hydrogels)

Chemically cross-linked hydrogels are formed by covalent linking ofthe chitosan macromers, where the bond formation is irreversible. Thecross-linking of natural and synthetic polymers can be achieved throughthe reaction of their functional groups (such as OH, COOH, and NH2)with cross-linkers (Liu et al., 2014; Milosavljević et al., 2010; Singhet al., 2006; Zhang et al., 2005). There are a number of methods re-ported in the literature to obtain chemically cross-linked permanenthydrogels. The following section reviews the major chemical methodsused to produce chitosan hydrogels.

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3.1.1. Chemical cross-linkersCross-linkers are molecules with at least two reactive functional

groups that allow the formation of bonds between polymeric chains. Todate, the most common cross-linkers used to obtain chitosan hydrogelsare di-aldehydes, such as glutaraldehyde (Milosavljević et al., 2010;Zhang et al., 2005), formaldehyde (Singh et al., 2006), or ethyleneglycol di-glycidyl ether (EGDE) (Liu et al., 2014), genipin (Mi et al.,2000; Muzzarelli, 2009b) and others (Draye, Delaey, Voorde, Bulcke,Bogdanov et al., 1998; Hennink & Nostrum, 2002; Huffman, 2012) (SeeFig. 2).

Among these mentioned cross-linkers, reaction of aldehydes withchitosan is well-documented; the aldehyde groups form covalent iminebonds with the amino groups of chitosan, due to the resonance estab-lished with adjacent double ethylenic bonds (Monteiro & Airoldi,1999). Di-aldehydes allow direct reaction in aqueous media, under mildconditions and without the addition of auxiliary molecules, such asreducers, which is advantageous with respect to biocompatibility.However, the main drawback of di-aldehydes, such as glutaraldehyde,is that they are generally considered to be toxic. It is reported thatglutaraldehyde is cytotoxic even at low doses and glutaraldehydepolymerization may release glutaraldehyde residues during storage orsterilization (Chang et al., 2004; MacDonald & Pepper, 1994). Glutar-aldehyde is known to be neurotoxic, its fate in the human body is notfully understood. Therefore, even if hydrogels are purified before ad-ministration, the presence of free unreacted di-aldehydes in hydrogelscould not be completely excluded and may induce toxic effects. Thiscould impair the biocompatibility of the cross-linked products (Berger,Reist, Mayer, Felt, Peppas et al., 2004; Chen et al., 2004).

In order to obtain non-soluble hydrogels for controlling drug releaseand pharmaceutical applications, researchers have been attempting tofind a cross-linking agent with low cytotoxicity (Chen et al., 2004; Cuiet al., 2014; Delmar & Bianco-Peled, 2015). Genipin is a natural cross-linking agent extracted from geniposide, an iridoid glucoside isolatedfrom the Genipa fruits (Tsai et al., 1994). Genipin has been reported tobind with biological tissues (Sung et al., 2001) and biopolymers, such aschitosan and gelatin (Bigi et al., 2003; Mi et al., 2005), leading tocovalent coupling. It works as an effective cross-linker for polymerscontaining amino groups (mostly used for chitosan crosslinking) (Sunget al., 1999). It has been found that the reaction rate of genipin withamino group containing biomaterials is significantly slower than that ofglutaraldehyde (Moura et al., 2007; Sung et al., 2000). The reactionbetween chitosan and genipin (See Fig. 3) is well understood for avariety of experimental conditions and has no cytotoxicity for humanand animal cells (Muzzarelli et al., 2015). Biocompatibility,

biodegradability and pharmaceutical effects of the genipin-crosslinkedhydrogels were investigated, and it was concluded that genipin may bewell suited for clinical usage (Kitano, 2006; Muzzarelli, 2009a; Yaoaet al., 2005; Zhang et al., 2006). Effects of genipin cross-linking ofchitosan hydrogels on cellular adhesion and viability were investigated.It was found that the cross-linked hydrogels did not induce cytotoxiceffects and significantly improved the cell adhesion and viability onhydrogel surfaces (Gao et al., 2014). Genipin has also been shown to actsignificantly as an anti-inflammatory and anti-angiogenesis agent, andprotected the hippocampal neurons from the toxicity of Alzheimer’samyloid beta protein (Kooa, 2004).

3.1.2. Photo crosslinkingPhoto-crosslinking, a type of chemical crosslinking, is performed in

the presence of ultraviolet (UV) light and a chemical photo initiator(PI). A variety of natural and synthetic macromers have been modifiedto formed hydrogels by photo activation of the coexisting photo in-itiators, such as methacrylated or aryl azide modified macromers(Rickett et al., 2011). The degree of crosslinking reaction depends onUV irradiation time and increases with the increase of exposure time.This lead to a hydrogel with higher mechanical properties and lowerswelling ratio (Ik et al., 2016). The polymerization reaction can alsocontrolled by adjusting the distance from the UV light source (Ahmadi,Oveisi, Mohammadi Samani, & Amoozgar, 2015). Obara et al.. (Obaraet al. (2003)) prepared a photo cross-linkable chitosan aqueous solutionincluding fibroblast growth factor-2 (FGF-2) by using ultraviolet light(UV) irradiation to result in an insoluble, flexible hydrogel. A viscousazide-chitosan-lactose aqueous solution was converted into an insolublehydrogel within 10 s upon UV-irradiation at a lamp distance of 2 cmthrough crosslinking of the azide and amino groups of the azide-chit-osan-lactose molecules. The results showed that the growth factor FGF-2 molecules remained biologically active, and were released from thechitosan hydrogel upon in vivo biodegradation. Application of thechitosan hydrogel significantly induced wound contraction and ac-celerated wound closure.

Among the photo initiators, Irgacure2959 caused minimum cyto-toxicity (cell death) over a broad range of mammalian cell types andspecies (Hu & Gao, 2008). Pluronic-chitosan hydrogels used as a dres-sing for ulcers wounds were fabricated, and the release of growth factorwas investigated. Photo-irradiation was applied to chemically cross-linkthe hydrogel. Before irradiation by UV, a photo-initiator, Irgacure2959(0.1%, w/w), was added to the hydrogel mixture to form physical hy-drogels. Release profiles of rhEGF growth factor from hydrogel net-works were evaluated with modification of the chitosan blend ratiosand 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 ofhydrophobic interactions. Increasing exposure time led to a tight net-work which can control growth factor release (Choi & Yoo, 2010b).

3.1.3. Graft polymerizationGraft 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 containingtwo types of reactive groups is of considerable interest for modificationof polymer structure. Chitosan has two types of reactive groups that canbe grafted; the free amine groups on deacetylated units and the

Fig. 2. Chemical Cross Linker Structures.

Fig. 3. Cross linking between Genipin and Chitosan (Muzzarelli et al., 2015).

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hydroxyl groups on the C3 and C6 carbons of acetylated or deacetylatedunits. Recently researchers have shown that chitosan derivatives, suchas hydroxypropyl chitosan (HPCT) and carboxymethyl chitosan sodium(CMCTS) functionalized by grafting, improved water solubility, anti-bacterial, and antioxidant properties (Alves & Manoa, 2008; Xie et al.,2002; Xie et al., 2001).

3.1.3.1. Chemical grafting. Chemical grafting is initiated by generatingone or more free radicals on the chitosan chain and allowing theseradicals to react with polymerizable monomers that build the graftedchain (Girin et al., 2012). In recent years, a number of initiators such asFerrous Ammonium Sulfate (FAS), Potassium PerSulfate (PPS), CericAmmonium Nitrate (CAN), and Ammonium PerSulfate (APS) have beendeveloped to initiate grafting copolymerization.

Yazdani-Pedram et al. (Yazdani-Pedram, Retuert, and Quijada,(2000)) modified chitosan by grafting with polyacrylic acid, a well-known hydrogel forming polymer. The reaction was carried out in ahomogeneous aqueous phase by using PPS and FAS as the redox in-itiators. It was observed that the efficiency of grafting depended onmonomer, initiator, and ferrous ion concentrations, as well as reactiontime and temperature. Also the level of grafting could be controlled byvarying the amount of ferrous ions used as a co-catalyst in the reaction.

Jung et al. (Jung, Chung, and Lee, (2006)) prepared a chitosan andeugenol hydrogel to enhance and maintain antioxidant activities byusing CAN as initiator. Eugenol is generally recognized as a safe ma-terial by the Food and Drug Administration, so it can be used in bio-logical applications.

The graft yield increased with the concentration of CAN. Thermalstabilities of chitosan alone and eugenol-chitosan hydrogels weremeasured using TGA analysis and it was observed that the eugenol-chitosan hydrogels have a faster thermal decomposition in comparisonwith that of chitosan alone. This is likely because the introduction ofthe bulky eugenol side chains inside the hydrogel decreased the crys-talline regions of chitosan. They observed that the equilibrium–swellingratio of eugenol-grafted chitosan hydrogels is smaller than that ofchitosan gels and decreased with increased graft yield, because of thehydrophobicity of the attached eugenol molecules. Because chitosan isa cationic polymer, the swelling ratio of chitosan hydrogels decreasedwith increasing pH values, but the swelling ratios of the more hydro-phobic eugenol-grafted chitosan hydrogels were not affected by the pHof the media, because chitosan amino groups, which were pH sensitive,were grafted with eugenol.

The pH responsive property of chitosan hydrogels can be explainedas indicated in Fig. 4 (Zou et al., 2015). At low pH, the amide groups onthe chitosan can become protonated and the resulting electrostatic re-pulsion between the protonated amino groups weaken the

intermolecular and intramolecular hydrogen bonding interaction ofchitosan molecules. Consequently, buffer solution can easily diffuseinto the network and lead to increased swelling ratios. In neutral andbasic conditions, no protonation occurs and the swelling ratio is low.

Mahdavinia et al. (Mahdavinia, Pourjavadi, Hossein zadeh, andZohuriaan, (2004)) introduced graft copolymerization of mixtures ofacrylic acid (AA) and acrylamide (AAm) onto chitosan by using PPS as afree radical initiator and methylene bis-acrylamide (MBA) as a crosslinker. It was concluded that the swelling capacity of the hydrogels isaffected by the MBA cross linker concentration and monomer ratio, sothat the swelling decreases with increased MBA concentration andAAm/AA ratio. They also concluded that this pH–sensitive super-absorbent poly-ampholytic network may be considered as an excellentcandidate to design influential drug delivery systems.

3.1.3.2. Radiation grafting. Grafting can also be initiated by the use ofhigh energy radiation from gamma rays and from electron beams.Radiation graft copolymerization is a well-established technique forproducing polymeric materials which combine chemical and physicalproperties of both base polymer and grafted monomer. Graftingcopolymerization of thermal and pH-sensitive chitosan and N-isopropyl acrylamide hydrogels were investigated by γ-radiation (Caiet al., 2005). In this research the effects of monomer concentration andirradiation dose on grafting percentage and efficiency were examined.Grafting percentage and efficiency increased with increased monomerconcentration and total irradiation dose. However, γ-radiation degradeschitosan. Sterilization of chitosan by γ-radiation leads to discolorationand loss of amino groups and lowering of molecular weight.

Compared to other methods, radiation crosslinking doesn’t requireany additives to start the process, so the final product contains onlypolymer. Moreover, ionizing radiation usually provides combination ofthe synthesis and sterilization of polymeric materials in a single tech-nological step, which leads to reduced cost and production time.Therefore, ionizing radiation or electron beam radiation methods are anexcellent tool in the fabrication of materials for biomedical applications(Zhao & Mitomo, 2008). A series of hydrogels were prepared frompolyvinyl alcohol (PVA) and carboxy methylated chitosan with electronbeam irradiation at room temperature. The mechanical properties anddegree of swelling improved substantially after adding chitosan into thePVA hydrogels. These blend hydrogels, even at low concentration ofchitosan (3 wt%), exhibited satisfactory antibacterial activity against E.coli (Zhao, 2003). In another study, N-maleoyl-chitosan was synthesizedby reaction of chitosan with maleic anhydride (MAH) in N,N-dimethylformamide (DMF) via electron beam irradiation. Grafting yield andgrafting efficiency increased with increasing absorbed radiation doseand monomer amount, and then decreased. Usually, the free radicals in

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

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the reaction system increase with dose and then eventually decay. Theswelling ratio of the copolymer hydrogel was low at pH 4–5 (Fan et al.,2009).

3.2. Physical Cross linking (reversible hydrogels)

Physically cross linked gels have attracted much interest recentlydue to the absence of chemical crosslinking agents and reagents. Manychemical crosslinking agents are toxic compounds which can be de-tached or isolated frequently from prepared gels before application.They can also affect the nature of the substances when entrapped (e.g.proteins, drugs, and cells) (Kamoun et al., 2015). The main dis-advantages of physical gels are their instability (uncontrolled dissolu-tion may occur), low mechanical resistance, and difficult control of poresize (Croisier & Jérôme, 2013). Also free chain ends or chain loops arealso present as transient network defects in physical gels (Huffman,2012). Some of the most common physical cross-linking methods forchitosan hydrogel preparation are now described.

3.2.1. Hydrophobic interactionPolymers with hydrophobic domains can crosslink in aqueous en-

vironments via reverse thermal gelation, also known as ‘sol-gel’ chem-istry. The hydrophobic segment is coupled to a hydrophilic polymersegment by post-polymerization grafting or by directly synthesizing ablock copolymer to create a polymer amphiphile. These amphiphilesare water soluble at low temperature. But by increasing the tempera-ture, hydrophobic domains aggregate to minimize the hydrophobicsurface area contacting the bulk water, reducing the amount of struc-tured water surrounding the hydrophobic domains, and maximizing thesolvent entropy. The temperature at which gelation occurs depends onthe concentration of the polymer, the length of the hydrophobic block,

and the chemical structure of the polymer (Hoare & Kohane, 2008).Tien et al. (Tiena et al., 2003) studied the N-acylation of chitosan

with fatty acyl chlorides to introduce hydrophobicity for use as a matrixfor drug delivery. They concluded that hydrophobic interactions en-hance the stability of substituted chitosan via hydrophobic self-as-sembly. Also release of drugs depended on the acyl chain length and thedegree of acylation and could be managed by diffusion, or by swellingfollowed by diffusion. An injectable in situ thermosensitive chitosan–β-glycerophosphate (CeGP) gel formulation has been proposed for tissuerepair and drug delivery (Ruel-Gariépya et al., 2002). The sol/geltransition can be summarized as a competition between several inter-molecular interactions: 1) the electrostatic repulsion between positivelycharged chitosan molecules, 2) a screening effect on this repulsion,induced by the negatively charged β-glycerophosphate, and 3) attrac-tive interactions due to hydrophobicity and hydrogen bonding betweenthe chitosan molecules (Supper et al., 2013). This system can releasemacromolecules over a period of several hours to a few days. The re-sults presented in this work indicated that the liposome–C–GP systemcould be used for the delivery of small molecular weight hydrophiliccompounds. The release profile of the incorporated compound can becontrolled by adjusting liposome characteristics, such as size andcomposition. This formulation can be a good candidate for local releaseof drugs and biomedical applications which require the local and con-trolled delivery of pharmaceuticals, such as growth factors in tissuerepair.

3.2.2. Polyelectrolyte complexationFormation of chitosan hydrogels by polyelectrolyte complexation

(PEC) has become an alternative for cross-linking. In the last decadethere has been an increasing interest in the use of PEC gels formed bychitosan and polyanions as carriers for drug delivery systems. PECs are

Fig. 5. Scheme of polyelectrolyte complexing reaction between chitosan and pectin (Archana, Dutta et al., 2013).

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generally biocompatible networks exhibiting interesting swellingcharacteristics. PEC gels formed by the electrostatic attractions betweentwo oppositely charged polyelectrolytes in aqueous solution are knownto exhibit unique physical and chemical properties. For instance, theelectrostatic interactions within the PEC gels are considerably strongerthan most secondary binding interactions. The electrostatic attractionbetween the cationic amino groups of chitosan and the anionic groupsof the other polyelectrolyte is the main interaction leading to the for-mation of PECs (Argin-Soysal et al., 2009; Berger, Reist, Mayer, Felt,Gurny et al., 2004). However, the main drawback of these systems istheir difficult preparation, especially in large-scale processes (Berger,Reist, Mayer, Felt, Gurny et al., 2004; Long & Luyen, 1996).

Archana et al. (Archana, Dutta, and Dutta, (2013)) have studied aternary nano-dressing consisting of titanium dioxide (TiO2) nano-particles loaded into a chitosan pectin polyelectrolyte complex that wasprepared to evaluate biocompatibility, and antimicrobial and in vivowound healing properties. The electrostatic attractions between theionized amino groups of chitosan (NH2) and the ionized carboxyl acidgroups (COO−) of pectin are the main interactions leading to the for-mation of the pectin/chitosan PECs illustrated in Fig. 5. The tensilestrength of the blended dressing material increased with decreasingpectin content (1:1) and incorporation of TiO2 nanoparticles. This nano-dressing exhibited superior antimicrobial activity against five patho-gens (E. coli, S. aereus, P. aeruginosa, B. subtilis and A. niger) and goodblood-compatible properties. The chitosan–pectin–TiO2 dressing mate-rial could control evaporative water loss from wound beds at an optimallevel and absorb more exudates, keeping the wound beds moist withoutrisking dehydration or accumulation of exudates. Also the nano-TiO2

particles in the chitosan pectin matrix have been shown to decreasecytotoxicity.

Structurally stable chitosan and γ-poly glutamic acid (γ-PGA) PEChydrogel formed through simple ionic complex interaction, between theamine groups of chitosan and the carboxylic acid groups of γ-PGA, wasconfirmed by FTIR spectroscopy (Tsao et al., 2010).. It was found thatthe chitosan–γ-PGA PEC hydrogels show antibacterial activity andpromoted cell proliferation. Therefore, the chitosan–γ-PGA polyelec-trolyte hydrogel appears to have potential as a new material for bio-medical applications.

3.2.3. Freeze-Thaw processingThe mechanical properties of chitosan hydrogels are not adequate

for some biomedical applications and many researchers have tried toimprove them. In order to achieve good mechanical and biologicalperformance, a combination of a synthetic and a natural polymer seemsto be a good choice. These materials are known in the literature as‘bioartificial’ polymeric materials. Among synthetic polymers, poly-vinyl alcohol (PVA) has been widely used in biomedical and bio-chemical applications because of its excellent chemical and physicalproperties, biodegradability, easy preparation and low cost. Blends ofchitosan with PVA have been reported to have good mechanical andchemical properties (Milosavljević et al., 2010).

Hydrogels prepared by freeze – thaw processing of poly vinyl al-cohol (PVA) solutions have drawn extensive attention. Some ad-vantages of physically cross-linked hydrogels obtained by freezethawing are the nontoxic, noncarcinogenic, and biocompatibility of theresulting polymers, good mechanical strength and the absence ofcrosslinking agents or initiators in the hydrogel synthesis (Ricciardiet al., 2004; Smith et al., 2009; Xiaomin et al., 2008). During thefreezing step, a liquid–liquid phase separation and formation of icecrystals in the polymer-poor phase occurs. Polymer chains in thepolymer-rich phase lead to the formation of hydrogen bonding and PVAcrystallites (See Fig. 6). In addition, the thawing procedure facilitatesthe interactions and formation of crystalline regions between the re-maining polymers, leading to formation of hydrogel networks(Holloway et al., 2013; Zhang et al., 2013). The ice crystals act as cross-linkers during the formation of hydrogels and leave porous structures in

the hydrogels, because of the space left from the melting ice crystalsduring the thawing stages (Guan et al., 2014).

The size of ice crystals formed in the structure increases with thenumber of repeated freeze/thaw cycles (Guan et al., 2014). Ricciardiet.al (Ricciardi et al., 2004) studied the structure of physical PVA hy-drogels prepared by freeze-thaw cycling with a systematic X-raypowder diffraction technique, as a function of the number of cycles andaging time. Their results showed that both the degree of crystallinity ofthe gels and the size of PVA crystallites increased with increasingnumber of freeze/thaw cycles as illustrated in Fig. 7. The increaseddegree of crystallinity is mostly due to the increase in the crystallitesize, although formation of new crystalline aggregates could not beexcluded.

Guanghua et al. (Guanghua et al. (2008)) prepared physically cross-linked PVA/chitosan hydrogels by the freeze/thaw technique. Theswelling results indicated that the hydrogels have good pH-sensitiveproperties in acidic environments and maximum swelling rate riseswith the increase of chitosan content in the blend, but leads to moredissolution at pH 1.0. Longer freeze/thaw cycle times result in a lowerswelling rate. They concluded that the swelling behavior of the com-posite hydrogels could be adjusted by changing the chitosan contentsand the freezing/thawing cycle times.

PVA hydrogels including chitosan were synthesized by combinedirradiation and freeze-thaw cycling and their properties were comparedwith those prepared by irradiation or freeze-thaw cycling alone. It wasfound that hydrogels made by irradiation alone have large swellingcapacity and are transparent, but too weak to be used as a wounddressing. Hydrogels made by irradiation followed by freeze-thaw havelarge swelling capacity, good thermal stability, high mechanicalstrength, and translucent appearance. All the hydrogels containingchitosan showed pH sensitivity and antibacterial activity (Yang, Liuet al., 2008).

Fig. 8 shows the effects of the hydrogel preparation method onmechanical compression ratio (Afshari et al., 2015). The mechanicalstrengths of the hydrogels prepared by the combinational method arehigher than that of the hydrogels prepared only by radiation. This is dueto the presence of physical crosslinks in addition to chemical crosslinks.This fact suggests that in order to increase the mechanical strength ofhydrogels prepared by radiation, the freeze-thawing treatment afterirradiation is very effective [Yang et al., 2008, 2010; Boynueğri et al.,2009].

4. Clinical studies

There are few prospective clinical trials in the area of chitosan hy-drogels for wound repair. Some clinical studies have been reported forusing chitosan in various medical fields [Diamond et al., 2003; Méthotet al., 2016; Mason & Clarke, 2015]. A research has been done on achitosan gelling fiber dressing (Kytocel®) in Staffordshire communitycare to examine improved healing of patients with chronic non-healingwounds. KytoCel® (Aspen Medical) is a highly absorbent dressingcomposed of chitosan fibers which bond with wound exudate to form aclear gel absorbing pathogens and is conformable to the wound bed.Over a 4-week period or until complete wound healing, leaking, stri-kethrough, pain, time, and malodour were examined. This new ad-vanced wound dressing has the ability to gel when in contact withwound fluid, making it less painful to remove, suitable for moderate tohigh exudate, reduced bioburden, and maintain haemostasis (Mason &Clarke, 2015). Another clinical study was conducted at three medicalcenters in China. A total of 90 patients with unhealed chronic woundsincluding pressure ulcers, vascular ulcers, diabetic foot ulcers, andwounds with minor infections, or at risk of infection, were treated withchitosan wound dressing or traditional Vaseline gauze as a control(Foshan United Medical Technologies Ltd, Guangdong, China). After 4weeks of treatment, the wound area reduction was significantly greaterin the test group than the control group. The average pain level in the

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test group was 1.12 ± 0.23 and 2.30 ± 0.23 in the control group. Themean duration of the test group was 27.31 ± 5.37 days and the controlgroup 27.09 ± 6.44 days. Consequently, the new chitosan wounddressing can enhance the wound healing process and reduce the pa-tient’s pain level. Furthermore the dressing was shown to be clinicallysafe and effective in the management of chronic wounds (Mo et al.,2015).

An evaluation of healing at split skin graft donor sites, dressed halfwith chitosan and half with a conventional dressing, revealed that

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

5. Chitosan application for drug delivery in wound dressings

Wound healing is a complicated process including four overlappingphases: coagulation, inflammation, migration-proliferation (includingmatrix deposition), and remodeling (Falanga, 2005; Kirsner &Eaglstein, 1993; Velnar et al., 2009). The human body can provide allthe requirements for the healing process in normal wounds, unless thereis any kind of deficiency of skin function or massive fluid losses in largewounds. However, providing appropriate pH, moisture, oxygen pres-sure, and preventing microbial invasion can accelerate the woundhealing process (Field & Kerstein, 1994; Guo & DiPietro, 2010). Woundcare for non-healing wounds has recently been a growing concern ofmany wound dressing applications involving various mechanisms, suchas coagulation, inflammation, angiogenesis, epithelization, contractionand remodeling. An ideal wound dressing should protect the woundfrom bacterial infection, provide a moist and healing environment, andbe 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 abilityto accelerate wound healing, it has attracted many researchers to beused as a wound dressing material. Applications of chitosan in differenttypes of wound dressing are explained in the following sections.

5.1. Application of growth factors in chitosan-based wound dressings

Growth factors are polypeptides which control the growth, differ-entiation, and metabolism of cells and regulate the process of tissuerepair. Despite their small amounts, they exert a powerful influence onthe process of wound repair. Use of growth factors for accelerating thehealing of wounds presents tremendous promise as a therapeutic ap-proach in treating chronic wounds. Several peptide growth factors, suchas epidermal growth factor (EGF), platelet derived growth factor(PDGF), fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β), have been shown to accelerate cellular proliferation andsynthesis of the extracellular matrix (Alemdaroğlu et al., 2006; Paul &

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

Fig. 7. Apparent dimensions of PVA hydrogel crystallites as a function offreeze/thaw cycles (Ricciardi et al., 2004).

Fig. 8. Comparison of mechanical strength of hydrogels prepared byCombinational and radiation method (Yang, Zhu et al., 2008).

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Sharma, 2004). EGF has been reported to increase the rates of cellularproliferation, growth and regeneration of tissue in numerous papers(Choi et al., 2012; Su et al., 2014; Yenilmez et al., 2015).

Regarding EGF and chitosan properties for wound healing,Alemdaroğlu et al. (Alemdaroğlu et al. (2006)) developed an effectivechitosan hydrogel formulation containing EGF to determine its effect onhealing of second-degree burn wounds in rats. Burns are classified ac-cording to the depth of the injury. In superficial second-degree burns,the epidermis and the superficial dermis are mainly affected. And thesekinds of burns are very painful. According to the in vitro release studies,the release of EGF from the chitosan gel was found to be 97.3% after24 h. The release kinetics from the gel formulation was found to be ofthe first order, and the release rate of EGF from the gel varied with time.The in vivo study exhibited significant increase in cell proliferation inthe EGF containing gel applied group. The average numbers of pro-liferating cells observed on the 3rd, 7th and 14th days are presented inTable 1. Evaluation of these immunehisto-chemically observed resultsshowed that the rate of healing in the EJ group was increased. Finally ithas concluded that this formulation can play an important role in burnwound treatment.

Park et al. (Park, Clark, Lichtensteiger, Jamison, and WagonerJohnson, (2009)) prepared chitosan scaffolds loaded with basic fibro-blast growth factor (bFGF) contained in gelatin micro-particles andtested for clinical relevance in an aged mouse model. The application ofthis kind of wound dressing was for accelerating the healing process ofpressure ulcers. bFGF was successfully delivered to the wound and in-creased angiogenesis compared to chitosan alone. They showed thatlevels of bFGF in wound tissue were significantly higher, indicatingsuccessful and prolonged growth factor delivery without burst effects.Finally they concluded that chitosan loaded with bFGF is a good can-didate for treatment of chronic wounds and reduces the proteolyticenvironment of the wound and potentiates bFGF activity, leading toaccelerated wound closure.

Table 2 Summarizes some reported chitosan hydrogels with in-corporated growth factors for wound dressing applications.

5.2. Nanoparticle and nanostructure incorporated hydrogels

Nanotechnology is developing as a new growing field with appli-cations in Science and Technology for the purpose of manufacturingnew materials at the nanoscale level. Nano-particles are used not onlyin fundamental chemistry and physics investigations, but also widely inbiomedical fields. Due to their good antibacterial properties, their largesurface area to volume ratios, and high microbial resistance, nanoscalematerials have emerged as novel antimicrobial agents (Sung et al.,2010; Radhakumary et al., 2011). Different types of metal materialshave been known to be used as antibacterial and/or/ antimicrobialagents in wound healing such as, silver, zinc oxide, titanium dioxide,copper oxide, and graphene. However, there could be problems withlong term exposure.

Sudheesh Kumar et al. (Kumbar et al., 2003) developed a flexibleand microporous chitosan/nano zinc oxide composite hydrogel. Atneutral pH, chitosan does not show much antibacterial activity; in orderto impart that activity, it is necessary to incorporate some antibacterialagents into it. Zinc oxide nanoparticles were used in this research due toits antibacterial activity. in vitro cytocompatibility studies revealed thatthe dressing showed enhanced cell viability and infiltration. in vivowound healing evaluation proved the healing ability and antibacterialpotential of the prepared hydrogel without causing toxicity to cells.They found that these advanced dressings can be used to prevent in-fections in burns and chronic and diabetic wounds. It is also possible tosynthesize chitosan quaternary salts that have a permanent cationicsite, at all pH.

Silver and silver ions have been known as effective antimicrobialagents for a long time. Owing to this ability, they have been applied to awide range of healthcare products, such as burn dressings, scaffolds,skin replacements and medical devices (Altiok et al., 2010).

Nano silver/gelatin/carboxymethyl (CM)-Chitosan hydrogels weresynthesized by radiation-induced reduction and crosslinking at ambienttemperature (Altiok et al., 2010). After incubation at 37 °C for 12 h,these hydrogels clearly showed antibacterial activity against gram-ne-gative E. coli. The hydrogel with 10mM nano silver content inhibitedmore than 99% of the E. coli. When the nano silver content decreased to5mM, the maximum inhibition ratio was around 50%, and when thenano silver content was further decreased to 2mM, the ratio decreasedto 26%. The results of this study suggest that nano silver/gelatin/CM-Chitosan hydrogels have potential as an antibacterial and anti-in-flammation wound dressing.

Nano-curcumin was incorporated in N,O-carboxymethyl chitosan/oxidized alginate hydrogel to develop a novel nano-composite hydrogelwound dressing (Gaysinsky et al., 2008). Curcumin, a natural productof the rhizomes of Curcuma longa, has been widely used as coloring

Table 1The average stained cell number in Chitosan gel with and without EGF(Yenilmez et al., 2015).

Groups The average stained cell number

3rd day 7th day 14th dayJ* 1.20 ± 0.17 2.43 ± 0.61 3.10 ± 0.68EJ* 1.63 ± 0.35 4.20 ± 0.18 4.95 ± 0.39

* Burn wounds treated by chitosan gel without (J) and with (EJ) with EGF.

Table 2Applications 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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agent and spice in food. But studies have shown that curcumin couldsignificantly accelerate the healing of wounds and enhance wound re-pair in diabetic impaired healing (Gaysinsky, 2007). Nano-curcumincould be released slowly from hydrogel to stimulate fibroblast pro-liferation, capillary formation and collagen production which sig-nificantly has effect on the healing phases. So, the nano-curcumin/chitosan/alginate hydrogel might have potential application in woundhealing.

Tigecycline nanoparticles were loaded into chitosan–platelet-richplasma hydrogel for wound infection treatment. The tigecycline nano-particle incorporated chitosan hydrogel showed a significant anti-bacterial activity against S. aureus. This system can be an effectivemedium for antibiotic delivery and applied on the infection sites toeffectively prevent various skin infections caused by S. aureus (Galottoet al., 2016).

Table 3 Summarizes some reported Chitosan hydrogels containingnanoparticles used for wound dressing applications.

5.3. Drug incorporated hydrogels

Controlled delivery systems provide an alternative approach toregulate bioavailability of therapeutic agents. In controlled drug de-livery systems, an active therapeutic is incorporated into a polymericnetwork structure, and the drug is released from the structure in apredefined manner. Depending on the drug delivery formulation andapplication, the drug release time may be from a few hours to severalmonths (Vimala et al., 2011). Different approaches have been devel-oped for drug incorporation. The method of drugs loading directlyimpacts the availability of the drugs during release.

According to the application of wound dressings and the types ofwounds, various kinds of drugs can be loaded in wound dressings. Sunget al. (Zhang et al., 2015) developed a minocycline-loaded wounddressing made with a PVA and Chitosan blended hydrogel made usingthe freeze-thaw method. Minocycline was used in this study because ithas been used in the treatment of superficial infections of the skin. Theminocycline-loaded wound dressing composed of 5% PVA, 0.75%chitosan and 0.25% drug was more swellable, flexible and elastic thanthe PVA gel alone, because of the relatively weak cross-linking

interaction of chitosan with PVA. According to their histologicaly ex-amined data presented in Table 4, hydrogels prepared with drug re-sulted in less inflammatory cells, more numerous collagen prolifera-tions, and microvessels in rats than the conventional product or thecontrol (sterile gauze). The healing effect of minocycline-loaded hy-drogel was great, indicating the potential healing effect of minocycline.Finally they concluded that the aforementioned minocycline-loadedwound dressing is a potential wound dressing with excellent formingand enhanced wound healing characteristics.

A thermo responsive and cytocompatible chitosan based hydrogelconsisting of thiolated chitosan with poly (N-isopropyl acrylamide)loaded with ciprofloxacin was introduced by Radhakumary et al(Mishra et al., 2017). They reported that polymers with thiol groupsprovide enhanced adhesive properties in comparison with many mu-coadhesive polymers, and the strong cohesive properties of thiolatedchitosan make it highly suitable for controlled drug release. Cipro-floxacin is a quinolone antibiotic drug recognized for treatment of skinand skin structure infections. Their prepared film exhibited appropriatemechanical properties for a wound/burn dressing and was found to becytocompatible and antibacterial as they released the entrapped anti-biotic in a controlled manner for a period of more than 48 h.

Hydrogel microspheres of polyacrylamide-grafted‐chitosan crosslinkedwith glutaraldehyde were prepared to encapsulate indomethacin, a non-steroidal anti‐inflammatory drug. The microspheres were produced by thewater/oil emulsion technique and encapsulation of indomethacin was car-ried out before crosslinking of the matrix. The initial release of in-domethacin is due to the polymer chain relaxation process, but at longertimes, the release occurs from the fully swollen polymer and is controlledmainly by the molecular diffusion phenomenon. This study suggests thatthe hydrogel microspheres prepared from chitosan‐based polymers can beuseful in the delivery of indomethacin‐like drugs (Zhang et al., 2018).

Additional drug-loaded chitosan hydrogels reported for wounddressing applications are presented in Table 5.

Essential oils and herbal extracts are natural complex mixtures ofvolatile, lipophilic substances obtained from different parts of plants bysteam distillation and solvent extraction. In recent decades, interest inessential oils for use in pharmaceutical and biomedical field has risen.Essential oils have been shown to possess antibacterial, antifungal,

Table 3Application 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.

Table 4Histomorphometrical 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/1mm2 of field) 4.40 ± 1.67 20.80 ± 6.72a 3.20 ± 2.28 23.40 ± 8.99Numbers of inflammatory cells (cells/1 mm2 of field) 2106.00 ± 934.45 250.00 ± 217.17a 2447.00 ± 527.81 179.00 ± 227.74Percentages of collagen tissue occupied regions (%/1mm2 of field) 36.73 ± 8.96 66.21 ± 10.07a 33.41 ± 4.50 75.81 ± 9.44

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antiviral, insecticidal and antioxidant properties due to their biologi-cally active compounds (See Table 5. for their applications in wounddressings). Chitosan films loaded with thyme oil were prepared by asolvent casting method (Jiang et al., 2016). Thyme oil has been shownto have inhibitory activities against various bacteria and yeasts (Shuklaet al., 2016). Thymol, the major component of thyme oil (Anjum et al.,2016), has also been shown to exhibit antimicrobial activity againstseveral bacteria and fungi (Koosehgol et al., 2017). The prepared filmsshowed both antibacterial and antioxidant activities. Results of thisresearch revealed that the thyme oil has a good potential to be in-corporated into chitosan to make antibacterial and permeable films forwound healing applications.

6. Conclusions

Wound healing is a complex and dynamic process of replacing de-vitalized and missing cellular structures and tissue layers. Many effortshave been focused on wound care with an emphasis on new therapeuticapproaches and development of technologies for wound management.In recent decades, smart wound dressings with accelerated woundhealing properties have attracted much interest.

This review summarized the potential applications of chitosan hy-drogels for pharmaceutical and biomedical uses, particularly with re-gard to drug delivery in wound dressings. Chitosan is an excellentcandidate due to its non-toxicity, stability, biodegradability, and bio-compatibility. Also chitosan hydrogel structures may be loaded withvarious types of drugs in different physical forms, such as nanoparticles,essential oils, and solubilized drugs. These properties have made chit-osan a very versatile material with extensive applications in controlledrelease formulations for treatment of acute and chronic wounds likeburns and pressure and diabetic ulcers. Therefore it can be concludedthat chitosan-based hydrogels are expected to become a promisingmatrix for use in regenerative medicine, drug delivery, and wounddressing applications.

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* EDC: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiiminde, NHS: N-hydroxysuccinimide, PEG: Poly ethylene glycol.NVP: 1-vinyl-2-pyrrolidone.

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