The physical and chemical properties of hydrogels based on natural polymers

CHAPTER 6

The physical and chemical properties of hydrogels based on natural polymers B. Kaczmarek*, K. Nadolna and A. Owczarek Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Nicolaus Copernicus University in Torun, Torun, Poland

6.1 Introduction Nowadays, hydrogels are a type of material which is of great interest to scientists working in a number of different fields. Hydrogels are three-dimensional polymeric networks which are able to bind water due to the hydrogen bond formation. They can be formed from molecules containing hydrophilic groups where the polymeric chains are in a colloidal state in the dispersion medium. Hydrogels have been defined in different ways. The most accurate is that hydrogels are water-swollen materials based on cross-linked polymeric chains. Another definition describes hydrogels as a type of material which exhibits a high ability to swell without changes to its structure, volume, or shape. Hydrogels can be flexible and easy to shape, depending on the application [1]. Such materials can be obtained from natural and/or synthetic polymers, which turn into gel according to various factors, for example, temperature, ionic strength, pH, and UV irradiation. A unique three-dimensional structure is formed due to the high hydrophilicity of polymers [2]. Hydrogels show swelling behavior dependent on environmental conditions, such as pH, temperature, ionic strength, and electromagnetic radiation [3]. Also, the properties of hydrogels including shape, mechanical flexibility, opacity, and porosity can be modified by changing these parameters. The water in hydrogels can be classified as water bound to the polymeric chain, free water, and semibound. When dry hydrogels are introduced into water, first water molecules penetrate the hydrogel and hydrate the hydrophilic groups (primary bound water). When groups are hydrated the polymeric network swells and exposes hydrophobic groups which interact with water molecules resulting in hydrophobically bound water molecules (secondary bound water). After the hydrophilic and hydrophobic sites of the hydrogel 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00006-9 © 2020 Elsevier Inc. All rights reserved.

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152 Chapter 6 interact with water molecules, the network imbibes additional water due to the osmotic forces of the polymeric network chains toward infinite dilution. Such additional swelling is resisted by chemical and physical cross-linking, resulting in the elastic hydrogel properties. The additional water which is absorbed by the hydrogel is called “bulk water” or “free water” and fills the spaces between the polymeric network chains [4]. Semibound water has intermediate properties between free water and those bound to the polymeric chain. Hydrogels can be divided into two main groups: based on synthetic polymers and natural polymers. Both types of hydrogel have advantages and disadvantages. However, considering the medical application of hydrogels, it can be assumed that natural-based materials are biocompatible, biodegradable, and nontoxic for the human body. Such features make them better materials than synthetic compounds. On the other hand, synthetic-based hydrogels have better mechanical parameters which enhances their potential industrial applications. Also, composite materials, which are a mixture of synthetic and natural polymers, are very common. There are different types of hydrogels: 1. Homopolymeric hydrogels—this network refers to a type of monomer, basic structural unit. They can have a cross-linked skeletal structure depending on the polymer and cross-linking type. 2. Copolymeric hydrogels—hydrogel is obtained from two or more monomer species, and one has to be hydrophilic. They can be arranged in various configurations. 3. Multipolymeric hydrogels—two independent cross-linked natural or synthetic polymers which form the network [5]. Hydrogels can also be classified depending on their charge (nonionic, ionic, amphoteric electrolyte, zwitterionic), physical appearance (matrix, film, or microsphere), based on the type of cross-linking (chemical or physical cross-linking methods), and the configuration (amorphous, semicrystalline, and crystalline). Natural polymer-based hydrogels recently replaced synthetic polymer-based materials, because they have several advantages. First, they are biocompatible and after application give excellent cellular and tissue responses. Proteins as well as polysaccharides can be used to design new hydrogels for tissue engineering purposes. Second, hydrogels obtained from natural polymers are biodegradable, and there is no formation of toxic products during the degradation process [6]. Hydrogel properties depend on the type of polymer, including the presence of hydrophilic groups attached to the polymeric chains ( OH, CONH , COOH, CONH2, SO3H). For instance, the presence of alcohol, amides, and carboxylic acids as hydrophilic moieties enhances hydrogel stiffness and the capacity to absorb water. Moreover, the presence of functional groups influences the type of cross-linking process needed for hydrogel formation.

The physical and chemical properties of hydrogels based on natural polymers 153 Hydrogels are a class of soft material with elastic properties. The low mechanical properties of hydrogels restricts their applications. Due to the presence of many functional groups they are easy to cross-link and as a result possess good mechanical strength [7]. Depending on the polymer properties used to obtain hydrogels and the density of the network, various amounts of water can be absorbed in the swollen state. Usually the mass of water is higher than the mass of polymer. Hydrogels are present in a variety of forms, such as slabs, membranes, beads, microgels, nanogels, cryogels, or aerogels. Hydrogels are very attractive materials due to their multifunctional properties which allow for the wide range of applications in different aspects of life, especially in biomedical science. They have the ability to absorb water and dissolved compounds, resulting in their application as napkins or superabsorbents used for water conservation. Hydrogels can be described as “smart materials” because they respond to various external factors including pH, temperature, and osmotic pressure. Such influences of external factors can find application in controlled-release systems in the fields of medicine or agriculture [8]. Hydrogels have found wide application in a variety of fields, such as agriculture, drug delivery, tissue engineering, water purification, contact lenses, and sensors [4].

6.2 Hydrogel preparation Hydrogels can be obtained in different chemical and physical ways. Hydrophilic and hydrophobic monomers are used to regulate hydrogel properties for specific applications. Hydrogels can be produced by reactive cross-linkers, copolymerization, or free-radical polymerization. Such material form can be obtained in a number of ways including ionization, linking polymer in a chemical reaction, or physical interactions. Bulk polymerization is the method used to produce hydrogels from one or more types of monomers. For the formulation a small amount of cross-linker has to be added. Then the initiator, as radiation, ultraviolet, or catalysts, is applied. The initiator used depends on the polymer structure and solvent nature. Such polymerization can be used to produce hydrogels including rods, particles, emulsions, membranes, and films. The other method, free-radical polymerization, can be applied to monomers as vinyl lactams, amides, or acrylates, with appropriate functional groups or which can be functionalized with radical polymerization. It includes the chemistry of typical free-radical polymerization steps, such as propagation, chain transfer, initiation, and termination. The free-radical method is based on monomers modifying into active forms. In solution polymerization the ionic or neutral monomers are mixed with cross-linking agents. The polymerization can be initiated thermally by UV irradiation or by the redox

154 Chapter 6 initiator system. In this method solvent (ethanol, water-ethanol, or benzyl alcohol) serves as a heat sink. The obtained hydrogels need to be washed in water to remove the initiator, unreacted monomers, oligomers, cross-linkers, and other impurities. Hydrogels based on natural polymers can be obtained by one-step polymerization, for example, in situ polymerization where the cross-linkers and nanoparticles can be incorporated. Such composites can find application as delivery systems, sensors, in tissue engineering, etc. [9]. Moreover, multiple-step polymerization can also be used to obtain hydrogels from polymers with reactive groups and their subsequent cross-linkers [5]. Many hydrogels are produced using hydrogen bond formation. The factors influencing the efficiency of this method are temperature, molar ratio of components, polymer structure, and the type of solvent.

6.3 Hydrogels from polysaccharides Polysaccharides are natural polymers constructed by repeating units of different saccharides bound together by glycosidic linkages. They range in structure from linear to highly branched. Polysaccharides can be extracted from different natural sources. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure, these macromolecules can have distinct properties from their monosaccharide building blocks. When all the monosaccharides in a polysaccharide are the same type, the polysaccharide is called a homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present they are called heteropolysaccharides or heteroglycans. Polysaccharides are widely used to obtain hydrogels due to the presence of hydrophilic functional groups which are able to absorb water and can be easily cross-linked by different chemical and physical methods. Hydrogels based on polysaccharides can be considered “injectable hydrogels” thanks to their ability to be squeezed through the needle of a syringe [6]. This review includes a discussion of hydrogel properties obtained from chitosan, alginate, carrageenan, hyaluronic acid, starch, and cellulose.

6.3.1 Chitosan-based hydrogels The main polysaccharide used to produce hydrogels is chitosan. Chitosan is a copolymer of β-(1-4)-linked 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose. It is a polycationic biopolymer which can be obtained by alkaline deacetylation from chitin, which is the main component of the exoskeleton of crustaceans. For any studies, the deacetylation degree and molecular weight of chitosan have to be detected because such factors have an influence on the polysaccharide properties. Chitosan is well known as a biocompatible

The physical and chemical properties of hydrogels based on natural polymers 155 compound which has found biomedical application in implantation, injection, and as a wound-dressing material. Other advantages include its low cost of isolation, its eco-friendliness, and nonhazardous nature. Chitosan contains amine ( NH2) and hydroxyl groups ( OH). Thereby it can be easily cross-linked to modify its properties. Hydrogels can be formed either from chitosan or from chitosan with other polymers. Chitosan is sensitive to pH changes and can be self-crosslinked by increasing the pH or dissolving in a nonsolvent [10]. There are a wide range of cross-linking methods which include the chain modification, use of the chemical reaction of chitosan and cross-linker, hydrophobic association, electrostatic interactions, or hydrogen bonding [11 14]. Also, chitosan-based hydrogels can be obtained by using physical methods such as UV-light irradiation [15]. Covalently cross-linked chitosan-based hydrogels can be divided into three groups—chitosan cross-linked by itself, hybrid polymer networks, and semi- or fullinterpenetrating polymer networks. Polysaccharide cross-linked by itself may involve two units from one or two different polymeric chains. The final structure has a gel network form. The hybrid chitosan network is formed between a polymeric chain of another polymer and chitosan structural unit [16]. In each cross-linking type covalent bonds are the main interactions, moreover secondary interactions, such as ionic, hydrophobic, and hydrogen bonds, can be formed. The modification processes influence the hydrogel properties, thereby appropriate characteristics of obtained materials can be obtained. Covalent cross-linking is based on the reaction between aldehyde and amine groups, where the imine bonds are formed. The use of aldehydes as chitosan cross-linkers is well-documented and studied. Other compounds which can be used as cross-linkers for covalent bond formation are diethyl squarate, oxalic acid, and genipin. However, the cross-linking mechanism is not wellknown, thereby cross-linked chitosan hydrogels by those agents have to be tested by an in vitro method for biocompatibility studies. The next method to form covalent cross-linking interactions is to add functional biopolymers, such as poly(ethylene glycol), cyclodextrin, and poly(vinyl alcohol). Nevertheless after such modification biocompatibility also needs to be studied. Chitosan hydrogels can also be obtained by complexation with collagen, poly(acrylic acid), chondroitin sulfate, and xylan. Chitosan was cross-linked with tris(2-(2-formylphenoxy)ethyl)amine. Hydrogels have been tested as drug-delivery systems and the results showed the pH and temperature-responsive swelling ratio [17]. Chitosan was also tested in the mixture with, for example, hyaluronic acid, carrageenan, and gelatin [18 20]. For instance, chitosan was tested in the mixture with poly(vinyl alcohol) and nano zinc oxide [21], where chitosan is used to improve the material biocompatibility. Polysaccharide was also cross-linked by genipin with poly(vinyl pyrrolidone) to from swellable hydrogels in different pHs and temperatures [22]. Chitosan was also mixed with cellulose and

156 Chapter 6 cross-linked by glutaraldehyde. The proposed hydrogels showed antimicrobial activity against Gram-positive and Gram-negative bacteria [23]. Chitosan can also form a hybrid composite with beta-glycerophosphate as injectable hydrogels able to mimic the extracellular matrix and provide a microenvironment for cell growth [24]. Chitosan-based hydrogels can also be obtained using an ionically cross-linking method. This avoids the purification and verification step. Chitosan is a positively charged polymer which can react with negatively charged components, ions, or molecules. As a result the ionic bridges between polymeric chains are formed. Mostly, the molecular weight of chitosan influences the cross-linking strength. The nature of the ionic cross-linking depends also on the type of cross-linker. The ammonium groups from the chitosan polymeric chain may interact with metallic ions to form coordinate-covalent bonds. Also, the positively charged ammonium groups may interacts with hydroxyl groups. Other hydrophobic or hydrogen interactions may also occur. The cross-linking density is an important factor which can be modified by the external conditions of the hydrogel preparation. The density influences the mechanical properties and swelling rate. The cross-linker agent structure, size and chemical character, temperature, pH, and solvent type may cause the hydrogel properties to change. The main influence on the ionic cross-linking is the pH of the solution. The acid conditions are necessary to form positively charged ammonium groups of chitosan, which then can be neutralized, for instance, in the dialysis process. The pH value during cross-linking must be in the vicinity of the pKa interval of the chitosan and cross-linker [25]. With high pH positively charged chains are neutralized. It can be assumed that the optimal pH for chitosan cross-linking is about 6. Novel materials based on chitosan contain microspheres or microcapsules with an active substances. They are attached to the polymeric network of hydrogels. Such composition improves skin penetration by the compounds, which enhances hydrogel application for skin moisturizing or regeneration. The important factor of such hydrogels is the pH, which must be appropriate to the pH value (4.9 5.6) of skin. Hydrogels were also prepared by in situ hydrogelation of chitosan biopolymer with nitrosalicylaldehyde in the presence of a model drug, varying the cross-linking density [26]. The injectable hydrogels can be formed from the chitosan and alginate mixture, where such hydrogel application results in an increase in the thickness and integrity of epidermal tissue, increased formation of collagen fibers, and enhanced expression of vascular endothelial growth factor as compared to the control group [27].

6.3.2 Alginate-based hydrogels Alginate is a hydrophilic polysaccharide linear binary copolymer composed of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues as monomers, constituting

The physical and chemical properties of hydrogels based on natural polymers 157 M-, G-, and MG-sequential blocks. Commercially available alginates are typically extracted from brown algae (Phaeophyceae), including Laminaria hyperborea, Laminaria digitata, Laminaria japonica, Ascophyllum nodosum, and Macrocystis pyrifera. Alginates can be also obtained by bacterial biosynthesis from Azotobacter and Pseudomonas [28]. Hydrogels based on alginate can be formed under very mild conditions, at room temperature, and in the absence of toxic solvents. They are ideal materials for biomedical applications, however, they need to be modified to resemble the mechanical, chemical, and structural properties. Alginate has wide applications to produce hydrogels due to its biocompatibility and biodegradability, gel-forming properties, and low costs. Alginate has groups easy to protonate, and in the pH above its pKa the polyanionic chains are formed, where the carboxylic groups are negatively charged. In such a form metallic ions including Ca21, Ba21, Cu21, Co21, Ni21, or Sr21 can cross-link the polymeric chain to form gel-like forms. Generally, alginate is hydrophilic and water-soluble, thickening in neutral conditions what can then be related to in situ hydrogel formation. Alginate-based hydrogels can be classified into physical and covalent gels. Various methods can be employed for hydrogel formation, such as ionic interaction, thermal gelation, cell-cross-linking, “click” reaction, and freeradical polymerization. Also, materials obtained from alginate have pH-responsive properties due to the presence of many carboxylic groups in the backbone [29]. Ionic cross-linking is the most common method to obtain hydrogels from an aqueous solution. The alginate structure block of M monomer forms weak junctions with divalent cations and the block of G monomers forms tightly held junctions. Different cations can be used for hydrogel preparation, however, calcium ions are most commonly used for alginate chains. Mainly CaCl2 is used for such purposes, however, the cross-linking is too fast to control. Therefore it can be replaced by CaSO4 or CaCO3, which have lower solubility [29]. The thermoresponsive phase transition can be proposed for alginate-based hydrogel formation simply as the temperature increase above the critical solution temperature. The thermosensitivity of an alginate hydrogel is achieved by incorporating, for example, poly(N-isopropylacrylamide) into its backbone [30]. The cell-cross-linking is a method which includes the specific receptor ligand interactions to cross-link the alginate-based hydrogels. The main strategy of this method is to introduce ligands, such as arginine-glycine-aspartic acid, onto the alginate for cell adhesion [29]. The formed network shows good biocompatibility, however, it has low strength and toughness. Another method, free-radical polymerization, is the process of transforming a linear polymer into a three-dimensional network. This depends on the pH and temperature as well as the addition of initiators.

158 Chapter 6 Alginate can be mixed with chitosan or hyaluronic acid to form novel biocompatible hydrogels [31]. Also, chondroitin sulfate was used as an additive to form hydrogel with alginate polymer, where the main influence on its properties was the molecular weight of the polymers [32]. Alginate-based hydrogels with gum tragacanthin were obtained by a gelation method followed by chitosan polyelectrolyte complexation and were tested as an oral insulin carrier. The proposed polymers are biodegradable, biocompatible, and nontoxic [33]. Alginate can be also mixed with synthetic polymers, for instance, poly(acrylic acid), and cross-linked by gamma irradiation [34]. Polysaccharide was also mixed with peptides, such as silk fibroin [35], collagen [36], and elastin [37]. The obtained materials presented significant biological properties.

6.3.3 Carrageenan-based hydrogels Carrageenan is known to possess multifarious groups including hydroxyl/sulfate groups. These are excellent for various chemical modifications, such as oxidation and carboxymethylation. They can be oversulfated, methylated, acetylated, and phosphorylated. Such modifications prove that carrageenan is a robust polymer and improve its physicochemical properties. Hydrogels based on carrageenan are generally formed through thermoreversible gelation, ionic cross-linking, as well as the photocross-linking of carrageenan backbone by methacrylate moieties [38]. Ionic cross-linking is a method which includes an interaction with K1 and Ca21 ions resulting in brittle hydrogels. Photocross-linking includes the incorporation of moieties such as methacrylate groups followed by UV cross-linking in the presence of photoinitatior (Irgacure 2959) where the hydroxyl groups are replaced by methacrylate moieties. Chemically cross-linked hydrogels had higher water retention capacity resulting in a more flexible network [38]. It can be assumed that higher metacrylation degree results in the lower swelling ratio and lower pore size of interconnected pores. Gradient hydrogels exhibit a gradual change in the viscoelasticity, stiffness, and porosity properties. They can be obtained by various methods, such as electrospinning, microfluidic, and gradient makers. A change in the cell morphology within the gradient hydrogel could be an indicator of the material’s potential to modulate the cell fate [38]. The hydrogels based on carrageenan have good gastroretentive properties. This extended gastroretention can be attained by utilizing the swelling property of the material which absorbs fluid from the surrounding environment, making it flow. It was assumed that the gel strength decreased with an increase in the concentration of the pore-forming agent, as more porous and fragile gel is formed. Hydrogels can be also obtained by the micropatterning method to design patterns in microscale for cell culture substitutes.

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6.3.4 Hyaluronic acid based hydrogels Hyaluronic acid is a nonsulfated polysaccharide that belongs to the glycosaminoglycans group. It is one of the main components of the extracellular matrix of many soft connective tissues. Hyaluronic acid is constructed by repeating units of D-glucuronic acid and N-acetyl-Dglucosamine, linked together via alternating β-1,4- and β-1,3-glycosidic bonds. Hyaluronic acid can be characterized by different molecular weights which influence its properties [39]. Hyaluronic acid is an attractive polymer for biomedical applications because it is biocompatible, biodegradable, nontoxic, bioactive, nonimmunogenic, and nonthrombogenic. It can form gels at high concentration solutions for high-molecular-weight hyaluronic acid which are viscoelastic and do not have longlasting mechanical integrity. It is necessary to modify it in chemical reactions or cross-linking processes. Moreover, additional biological active compounds can be added, for instance, cytokines and drugs. Hyaluronic acid based hydrogels are networks consisting of interconnected chains. Over the past few years researchers have created a wide range of hydrogels based on hyaluronic acid with increasing complexity and diverse functions [40]. Materials based on unmodified hyaluronic acid chains are not useful as biomaterials due to their susceptibility to degradation and inferior mechanical properties. To improve the functions covalent cross-linking has to be carried out. Hyaluronic acid has two functional groups ( COOH and OH) which influence the cross-linker type used for material modification. The carboxylic group can be modified by, for instance, addition of N-hydroxysuccinimide, dicycloheyl carbodiimide, or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The hydroxyl group can be cross-linked by, for example, glutaraldehyde, cyanogen bromide, octeylsuccinic anhydride, and methacrylic anhydride. Compared to native hyaluronic acid, the cross-linked type exhibits more robust mechanical properties and is less susceptible to enzymatic degradation [25,39]. However, the covalent cross-linking method requires toxic reagents which are not suitable for cells and tissues. For tissue engineering purposes the gelation kinetics should be fast enough to allow for cell encapsulation to the cell/gel material.

6.3.5 Starch-based hydrogels Starch is the most abundant storage polysaccharide in plants, as granules in the chloroplast of green leaves and in the amyloplast of seeds, pulses, and tubers. It can be isolated from different sources including corn, potato, tapioca, and wheat. Starch is constructed by a number of monosaccharides or glucose molecules joined together with α-D-(1 4) and/or α-D-(1 6) linkages [4]. Starch includes two main structural components—amylose and amylopectin. The proportions of these components depend on the extraction method and starch source, which affects the crystallinity and molecular order.

160 Chapter 6 Starch changes to gelatin in thermally three-step-assisted hydration-plasticization of the polymeric network. First, swelling is observed by absorbing water molecules. In the second step the gelatinization takes place after the starch dissolves by heating. This results in leaching of the amylose and causes irreversible physical changes. The granulate structure is destroyed. The final and third step is called the retrogradation step, in which the starch hydrogel network is created upon cooling and aging, result in recrystallization and reorganization of the polysaccharide structure [4]. The amylase content and temperature are two important factors influencing the process parameters and obtained hydrogel properties. There are different methods to modify the properties of starch. Starch has hydroxyl groups which can be utilized easily to prepare hydrogels. These modifications can be obtained by chemical reactions by introducing a small amount of ionic or hydrophobic groups into the starch chain. This results in a change to the solution viscosity and association behaviors. Esterified and grafted starches have been proposed to obtain hydrogels. In the etherification process some hydroxyl groups are substituted by ether groups. Also, various vinyl monomers can be grafted onto starch, such as acrylamide and acrylic acid [4]. Starch-based hydrogels have advantages and disadvantages. They are abundant natural biopolymers, biodegradable, with high availability, a renewable resource, economically attractive, easy to prepare, with high swelling capacity. However, they have low surface area and require chemical derivatization to enhance their sorption capacities. Interest in starch as a potential polymer to replace synthetic macromolecules has grown recently. There are two methods to obtain starch-based hydrogels. The first is to graft copolymerization of vinyl monomers on polysaccharides in the presence of a cross-linker. The second is the direct cross-linking of starch. Graft copolymerization begins with the starch reaction with the initiator. Such complexes are dissociated to create carbon radicals on the starch chain. The produced free radicals initiate the graft polymerization of vinyl monomers and cross-linker on the polysaccharide chains [4]. In the other method, the initiator abstracts hydrogen radicals from the hydroxyl groups of the starch to form initiating radicals of the polysaccharide chain. This reaction is influenced by temperature due to the use of a thermal initiator. Starch-based hydrogels have found application in dye removal from industrial wastewaters. Such hydrogels are preferred due to the availability of different adsorbents, their high efficiency, and easy handling. Different factors influence dye sorption, such as dye sorbent interaction, particle size, sorbent surface area, pH, temperature, and contact time. Starchbased hydrogels can be also used to remove heavy metals, which are harmful to humans, plants, and animals. Adsorption is an effective method for removal of heavy metal ions from aqueous solutions. They can also be used as superabsorbent hydrogels to decrease irrigation water consumption, enhance fertilizer retention in the soil, decrease plant death rates, and increase plant growth rates. In agriculture applications, hydrogels can also be

The physical and chemical properties of hydrogels based on natural polymers 161 applied in controlled pesticide formulations that reduce environmental pollution. Hydrogels may deliver pesticides slowly with the purpose of limiting their quantities. Advantages also include decreased leaching, degradation and volatilization, and the reduction of food residues and dermal toxicity.

6.3.6 Cellulose-based hydrogels Cellulose is a natural polymer present in a wide variety of living species including plants, animals, and some bacteria [3]. The most commercial source of cellulose is wood and plant fibers (cotton, jute, flax, etc.). It can also be produced by bacteria, algae, and marine animals. Cellulose exhibits good mechanical properties, and is biodegradable and hydrophilic. Moreover, it is biocompatible and, thereby, it is proposed for biomedical applications. Cellulose is constructed by repeating units of two anhydroglucose units (AGU) linked together through an oxygen covalently bonded to C1 of one glucose ring and C4 of the adjoining ring, β(1-4) glycosidic bond. The hydroxyl groups have high ability to form hydrogen bonds and play a major role in the properties of cellulose-based hydrogels. The intra- and intermolecular hydrogen bonds cause parallel arrangement of cellulose chains, which forms microfibrils. However, cellulose is insoluble in water and most organic solvents [3]. There are different methods to modify the properties of cellulose. Chemical modification includes esterification or etherification causing the obtainment of water-soluble cellulose derivatives. To regulate the solubility and viscosity in water solutions the degree of substitution can be controlled. It is defined as the average number of esterified/etherified hydroxyl groups from AGU [3]. Cellulose derivatives, such as methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), ethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, cellulose sulfate, and cellulose phosphate have been used to obtain reversible or stable hydrogels. Hydrogels can be obtained from cellulose and can form 3D networks. An important feature of hydrogels is their ability to swell and remain insoluble at the same time. Cellulose has excellent biocompatibility and is biodegradable [41]. Hydrogels based on cellulose can be obtained in chemical or physical cross-linking processes. Physical cross-linking is beneficial because it avoids the use of chemical cross-linking agents. It includes physical interactions such as chain entanglements, van der Waals forces, hydrogen bonds, and hydrophobic or electronic associations [3]. Hydrogel preparation can be carried out in a homogeneous medium, where cellulose is solubilized, and heterogeneous, where cellulose fibers are used to modify the mechanical properties of hydrogels. Cellulose can be dissolved in a few solvents, such as lithium chloride, N-methylmorpholine-N-oxide, ionic liquids, alkali

162 Chapter 6 aqueous systems, and alkali/urea aqueous systems. Hydrogels can be also obtained from cellulose nanoparticles, as nanofibrillated cellulose and cellulose nanocrystals. They can be also produced from bacterial cellulose, which has numerous advantages compared to plantderived cellulose, such as surface area, wet tensile strength, purity, crystallinity, and high biocompatibility [3]. Obtaining hydrogel is also possible by chemical cross-linking reactions. Cross-linking agents can be classified into two groups: esterifying agents where the formation of COOR bonds is observed and etherifying ones which result in R-O-R group formation. The most widely used cross-linking agent for polysaccharide chains is epichlorohydrin [42] and poly(vinyl alcohol) [43]. The swelling properties of obtained hydrogels depend on the cellulose/cross-linker ratio. For epichlorohydrin it increases with an increasing amount of cross-linker, which bucks the classical hydrogel trends. Also, hydrogels based on cellulosexanthan can be obtained. An increase in xanthan concentration permits an increase in the swelling of hydrogels [3]. Cellulose-based hydrogels can be obtained by radical polymerization of water-soluble cellulose derivatives with polymerizable groups [44]. Such hydrogels are characterized by high mechanical strength and stiffness, but also flexibility under different loads. Radicals can be formatted in situ of hydrogels by UV or visible light application in the presence of a photoinitiator. In the presence of hydrogel precursors bearing polymerizable groups, such as acrylate or methacrylate moieties, these formed hydrogels. The carboxymethyl cellulose (CMC)-methacrylate hydrogels present the controllable degradation rate, degree of swelling, and mechanical properties. Chemical cross-linking can be carried out with the use of epichlorohydrin, aldehydes and aldehyde-based reagents, urea derivatives, carbodiimides, and multifunctional carboxylic acids [3]. Cellulose can be mixed with chitosan and cross-linked by ethylene glycol diglycidyl ether [45], with sodium alginate, NaOH/urea solvent, and epichlorohydrin as cross-linker [46], with lignin in the presence of epichlorohydrin where the swelling ability is increased with an increasing lignin content [47]. Hydrogels with microfibrillated cellulose (MFC) incorporated in the alginate-based matrix cross-linked by pyrrole monomer by in situ polymerization were tested. The mechanical parameters were improved with a higher amount of TOMFC. Moreover, they were biocompatible and exhibited tunable swelling properties [48]. HPMC and sodium alginate were cross-linked by calcium ions [49]. The most used cellulose derivative as a natural component for hydrogels, due to its water solubility, low-cost, nontoxicity, and environmental friendliness, is CMC. The addition of CMC to cellulose in the NaOH/urea solution with cross-linker as epichlorohydrin contributed to the enhanced size of the pore, whereas cellulose was a strong backbone in the hydrogel to support it in retaining its appearance. Moreover, in different physiological fluids the hydrogels exhibited smart swelling and shrinking, as well as the release behavior of bovine serum albumin that could be controlled by changing CMC content [3].

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6.4 Agarose-based hydrogels Agarose-based materials can be projected and obtained as hydrogels, injectable hydrogels, self-healing hydrogels, scaffolds, and fibers. Agarose was also combined with other compounds, as well as its derivatives and blends, which have been already proposed. Agarose-based materials found application in tissue engineering, neurogenesis, cartilage formation spermatogenesis, wound healing, and as an artificial pancreas [50]. Agarose (copolymer of 3-linked β-D-galactopyranose and 4-linked 3,6-anhydro-α,Lgalactopyranose residues) is biocompatible polysaccharide which can be extracted from marine red algae. It can be then used to obtain thermal-reversible gel. Agarose can be also isolated from agar and its properties depend on the molecular weight of polymer. Agarose is polysaccharide which has high capacity to absorb water. It has a similar structure to the extracellular matrix, what supports cell proliferation and adhesion. Moreover, agarose has ionizable groups what result in the pH-responsive properties of polymer. The gelation process occurs in three steps, induction, gelation, and pseudoequilibrium, where the hydrogen bonds and other interactions form gel. The gelation process can be investigated by rheological methods and electron microscopy. Cross-linking can then be carried out without the need for a cross-linking agent in addition to the agarose. Hydrogels based on agarose can be obtained by agarose modification through a homogeneous reaction with acrylic monomers, and then by radical copolymerization [51]. The properties of hydrogels were modified by the addition of xanthan and resulted in the prevention of agarose gel aggregate formation [52]. The proposed hydrogels showed softer elasticity and a strong contribution to water molecules. The main factors influencing cross-linked materials are cross-linker type and concentration. Agarose-based hydrogels can be used as a cell immobilization matrix, drug-delivery system, or dental impression material.

6.5 Hydrogels from proteins Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. Proteins can be isolated from natural sources. They cannot be synthesized due to their complicated structure. Proteins are widely used to obtain hydrogels. They have hydrophilic groups present in the polymeric chain which can bind water molecules. Protein-based hydrogels have high ability to absorb water, which is beneficial for application possibilities. In this review hydrogels based on silk, keratin, and collagen are discussed.

164 Chapter 6

6.5.1 Silk-based hydrogels Silk is a natural protein present in the glands of silk-producing arthropods (such as silkworms, scorpions, mites, and bees) and spun into fibers. Silk for commercial use is isolated from silkworm silk, and is mainly produced by Bombyx mori [53]. Silk fibroin has several advantages over other proteins. The isolation process of silk is economically advantageous because silk is an established textile fiber. Silk fibroin can be isolated from wastes produced during silk fiber processing. Also, the purification process is carried out by simple methods, which makes silk available on a large scale. Silk-based hydrogels are eco-friendly and suitable to form biomaterials using different approaches [54]. They are currently receiving a great deal of interest for drug release due to their easy transformation from solution to gel form [55]. Silk-based hydrogels are formed through sol gel transition of aqueous silk solution. However, the sol gel transition time of a silk aqueous solution is long, usually a week or a month, which limits its practical use [56], therefore chemical and/or physical methods need to be applied to stimulate and enhance the gelation kinetics. Hydrogel formation can be carried out by different methods, such as organic solvent introduction, ultrasonification, vortexing, and pH changes. It has been illustrated that gel transformation of silk fibroin is possible by in situ gelation at a temperature 37 C due to self-assembly between the two different silk proteins [57]. Sol gel transition can be accelerated by increasing the temperature, protein concentration, and calcium ion addition. Silk fibroin forms a β-sheet structure which exhibits slow degradation in vitro and in vivo. It has to be cross-linked to improved its degradability and mechanical strength. In recent years silk-based hydrogels have been cross-linked by genipin and glutaraldehyde addition, and ionizing irradiation. Also, enzymatic methods have been applied including tyrosine [58]. Ionizing radiation is a method of hydrogel modification which uses gamma rays, electron beams, or ion beams. It result in radical formation on unsaturated polymer chains and water molecules, inducing intermolecular cross-linking. This proposed method eliminates the need for toxic cross-linking agent addition and is safe for the hydrogel preparation. Hydrogels from silk can have injectable and noninjectable forms. Inorganic particles can be incorporated into a three-dimensional hydrogel structure which improves their bioactivity [59,60]. Rapid cross-linking silk fibroin with surrounding tissue by an in situ method has attracted special attention and enhanced the application possibilities. Hydrogels based on silk fibroin were proposed as a drug-delivery system [55] or for the treatment of burn wounds [57].

The physical and chemical properties of hydrogels based on natural polymers 165

6.5.2 Keratin-based hydrogels Keratin is a fibrous protein which can be classified into two groups: soft keratin and hard keratin. It forms the bulk of cytoplasmic epithelia and epidermal structures. Keratin is abundant in animal hair, nails, wool, horns, and other features [8]. It may present in two conformations, α-helix and β-sheet. Compared to other proteins, keratin-based materials have higher stability and are not degraded by enzymes. Materials are mainly obtained from keratin isolated from wool and human hair [8]. The main source is wool, however, human-derivative keratin is of interest to researchers due to the reduced risk of an immune response. Keratin can be used in the synthesis of scaffolds for long-term cell culture. In hydrogels, keratin allows the formation of porous gel-form materials and creates a suitable environment for cell proliferation. They can be injected into a nerve conduit as a filter to guide nerve regeneration. Keratin-based materials are characterized by high strength and stability in in vivo conditions. They also accelerate cell regeneration and form compatible scaffolds. Keratin hydrogels can be easily rehydrated, which is desirable for invasive injection in prosthetics [61]. The properties of keratin-based hydrogels with collagen-based hydrogels cross-linked by the same cross-linker as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide have been compared [62]. It was assumed that keratin-based materials had a higher storage modulus and loss modulus. Also, the mechanical parameters for keratin without cross-linking were several-fold higher than those for cross-linked collagen. Generally, keratin-based hydrogels have appropriate features for biomedical purposes. Hydrogels obtained from keratin are relatively stable and have good mechanical strength, even without cross-linking modification. Also, the rehydrating methods are fast and easy, and very useful for commercial applications. It is possible to extend the storage of hydrogels by lyophilization. However, further studies of keratin-based hydrogels are needed.

6.5.3 Collagen-based hydrogels Collagen is the main protein in human connective tissues, accounting for 25% 33% of total proteins. It is constructed from repeating peptides (mainly proline, hydroxyproline, and glycine). There are 29 known types of collagen. Collagen I is a fibrous protein which is constructed of three α-chains intertwisting in a right-handed triple helix, stabilized by hydrogen bonds. Type I has been widely used to synthesize biomaterials, due to its abundance and well-understood structure from nano- to microscale. Collagen can be easily extracted from natural sources such as bovine skin or eyeballs, rat tail tendons, and fish scales.

166 Chapter 6 Collagen is widely used to obtain biocompatible, biodegradable, and safe materials including scaffolds, thin films, and hydrogels. The meat industry produces many by-products which contain collagen which can be extracted for further applications. The degree of cross-linking and molecular weight of collagen present in native tissues are significantly across the species. The source of collagen influences its thermal stability, mechanical parameters, solubility, and rheological properties [84]. Such factors play an important role in collagen-based material properties. The main sources of collagen include bovine hide, pig skin, shellfish, fish skin, and rat tendons. Most collagen-based hydrogels are prepared using type I collagen, and are ultimately formed in the presence of a water-based solvent. Along with the collagen content, the temperature of polymerization affects the hydrogel properties. Self-assembly of collagen molecules occurs more rapidly in higher temperatures, resulting in a less ordered structure. The room temperature is not uncommonly used but inhibits the inclusion of cells within hydrogel. Mostly previous studies have used a temperature of polymerization at around 37 C to facilitate cell seeding and viability [63]. Collagen-based hydrogels are sensitive to pH during the fabrication. This influences the structural and mechanical properties of collagen hydrogels. Mostly, hydrogels are formed with pH ranging from 5 to 10 and there is a strong positive correlation between pH and compressive modulus. The pH of collagen hydrogels depends on the type of buffer, the ratio of neutralization agent to acid-solubilized collagen, dilution ratio of collagen in hydrogel, and the soluble collagen concentration. Moreover, the ionic strength affects the collagen-based hydrogels polymerization. It also influences the mechanical and structural properties. Ionic strength is seldom measured directly for collagen hydrogels, rather it is calculated from known or estimated concentrations of all ionic compounds present in the solution [63]. Solubilized collagen must be storage at low pH and low temperature to prevent annealing of dissolved fragments. Hydrogels from collagen can be formed without adding any crosslinker. Fragments of dissolved collagen may aggregate and covalently bond together to reform fibrils in higher temperatures and pH. However, obtained hydrogels have weak mechanical properties, therefore cross-linker addition is necessary to enhance the mechanical strength of collagen-based hydrogels. Chemical cross-linking may also result in the improvement of resistance to degradation. The toxicity and safety of the cross-linking agent have to be considered. This is the reason more attention is now paid to natural cross-linkers [8]. Collagen implanted into the body can be recognized and bound by cells through integrin receptors, degrade collagen via specific enzymes, and synthesize new collagen. It is an important mechanism for tissue reconstruction to decide on the width of collagen application in medicine. However, collagen-based materials are characterized by low mechanical properties and rapid degradation rate, which limit their application [8].

The physical and chemical properties of hydrogels based on natural polymers 167 The kinetics of collagen fibril network assembly is a multistep process which includes fibril formation, fiber nucleation and development, and cross-linking, where the addition of external cross-linking agents affects fibrillogenesis [63]. For polymerization the ideal temperature and pH values have to be considered. Fibrillogenesis can be detected by spectrophotometric methods and allows determination of the degree of polymerization. The mechanical properties of collagen-based hydrogels have to be detected which implies matching both deformation modality and temporal characteristics. The viscoelastic properties can be measured by the deformation modalities including tension, compression, and torsion. However, it is difficult to assume that hydrogels have appropriate parameters for application in tissues, because tissues are in a state of constant change related to the physiological conditions. The settings and conditions of measurement can affect the obtained results. Hydrogels should maintain sample integrity by humidification or immersion in a buffer during measurement. Moreover, the collagen source and fabrication parameters influence the mechanical properties of obtained hydrogels. Recent studies have demonstrated the positive correlation between the collagen concentration and the elastic properties [64 66]. Also, increasing pH increases collagen-based hydrogel modulus [66 68]. The pore size of hydrogels determines the mechanical parameters. The addition of cross-linking agents increases the mechanical parameters of hydrogels as a result of new interactions/bond formation [69 71]. The fiber structure of extracellular matrices is an important quality of hydrogels because it can regulate their cellular morphology, migration, gene expression, and proliferation [70]. Parameters such as fiber diameter, density, and orientation influence material properties. Collagen fibers can be measured by different methods including atomic force microscopy, transmission electron microscopy, or scanning electron microscopy (SEM). Moreover, more advanced techniques can be used, such as cryo-SEM and environmental SEM, two-photon fluorescence, second harmonic generation, as well as confocal reflectance and fluorescence microscopy [72]. Collagen has been grafted to other polymers to form hybrid hydrogels, such as cellulose [73], chitosan [74], alginate [75], hydroxyapatite [44], and silver nanoparticles [76]. Irradiation cross-linking does not require any additives to start the process and the final product contains only polymer. Such a method modifies the hydrogel properties and can also act as a sterilizer. Thereby a one-step process reduces costs and production time. It is a tool in the fabrication of materials for biomedical applications [3].

6.6 Other natural polymers: lignin-based hydrogels Lignin is a phenolic polymer built up by three units: syringyl alcohol (S), guaiacyl alcohol (G), and ρ-hydroxyl alcohol (H). It is the second most abundant biopolymer. Lignin is a

168 Chapter 6 biocompatible, cheap, eco-friendly, and readily accessible material. Moreover, it is a biorenewable material and is available in large amounts as a by-product of forestry. Lignin shows also antimicrobial activities, enabling its biomedical applications. Lignin can be used in different fields due to its dispersing, binding, complexing, and emulsion-stabilizing properties. It can be used as an additive to animal feed, mesoporous materials, catalysts, or phenoplast glues. It can be also used to form organic hydrogels [77]. Hydrogels can be also obtained from lignin, which is a natural polymer, and is biorenewable and produced as a by-product of the forest industry [78]. They can be crosslinked by, for example, poly(methyl vinyl ether-co-maleic acid) [78]. Hydrogels were formed from lignin, with starch and hemicellulose by reactive extrusion [79]. Polymers were cross-linked by citric acid in the presence of a catalyst. The swelling behavior of the obtained hydrogels is highly dependent on the pH of the medium, where it increases at high pH values [79]. Other research includes hydrogel preparation from lignin with poly(ethylene glycol) and poly(methyl vinyl ether-co-maleic acid) through an esterification reaction [80].

6.7 Conclusion Hydrogels are widely studied materials for different industrial applications. They can be obtained from natural or synthetic polymers, which have hydrophilic groups. They have a high ability to swell. Nowadays, many natural polymers from polysaccharide and protein groups have been used for hydrogel preparation. Hydrogels can be obtained from pure polymeric chains by changing the temperature, pH, or solution. However, they are generally obtained in the cross-linking process. The type of polymer and modification procedure influence the hydrogel properties. Therefore it is necessary to detect their properties, such as mechanical parameters, thermal stability, degradation rate, and swelling behavior. It cannot be assumed which polymer is the best for hydrogel preparation, as each has advantages and disadvantages. Research into hydrogel materials is still in progress and are one of the most studied nanomaterial areas.

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