Hydrogels for Wound Healing Applications

Hydrogels for Wound Healing Applications Abstract This chapter discusses about the hydrogels both natural and synthetic that can be used for wound healing applications. Also it discusses briefly about the various novel techniques that have been developed recently. Keywords: Hydrogels; Chitosan, Antimicrobial; Grafting; Blending; Wound dressing; Wound healing; Gene therapy, Stem cell therapy, Skin Engineering, pH and Thermosensitive polymers. 1 Introduction Healthcare is an essential aspect of human survival. So many biopolymers have generated interest in a number of biomedical applications. Wound management is one such area where management of cuts, ulcers, and sores needs dressings which help in promoting rapid wound healing in order to obtain both functional and cosmetic results. [45] There are different kinds of wound management products: staples or sutures, dressings or bandages, surgical sealants and adhesives, skin substitutes, and other biomaterials. [51] Human skin provides an effective barrier to microbial penetration and subsequent infection. However, once the wound has been developed in this barrier, the infection chances increases. In case of chronic wounds, the colonization and infection potential increases as the result of the presence of avascular eschar which provides an environment for the uninhibited growth of microorganisms. [89] The rate of infection is related to the type of wound, general wound care, and local health of the patient. [88, 90] For avoiding infection, good clinical practices are needed. The management of chronic wounds is a very costly practice and it also places an enormous drain on healthcare resources; studies have calculated the cost of wounds to the NHS to be about  £1bn a year. [166] So for lowering this cost such wound management products are needed that are more economical and effective. Out of all the above wound management products, here in this chapter we will discuss more about the wound dressings that will provide an optimal healing environment to the wound. A dressing is an adjunct used by a person for application to a wound in order to promote healing and/or prevent further harm. It is designed to be in direct contact with the wound, so it is different from the bandage in the manner that bandages are normally used to hold dressing in place. A wound is a break in the epithelial integrity of the skin and may be accompanied by disruption of the structure and function of underlying normal tissue. Wounds can be divided into four categories based on their appearance and stage of healing: Necrotic, sloughing, granulating and epithelializing wounds. [87] Wounds cause discomfort and are more prone to infection and other troublesome complications. [75] Some diseases like diabetes, ischaemia and conditions like malnourishment, ageing, local infection, local tissue damage due to burn leads to delay in wound healing. Infection is a major complication of burn injury and is responsible for 50-75% of hospital deaths. [76] Human skin has one of the greatest capacities to regenerate itself amongst all of the tissues in our body. It constantly replaces old cells with new cells, enabling it to repair itself when damaged. Wound healing is a complex-physiologic process, which consists of three overlapping phases: inflammatory, proliferative and remodeling phases. The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors. During the inflammation process, neutrophils are the first leukocytes which come at the site of injury to rid it from bacterial contamination. Then, the monocytes and their conversion to macrophages initiate tissue repair by releasing a number of biologically active substances and growth factors that are necessary for the initi ation of tissue formation process. In the third process, fibroblasts proliferate and migrate into the wound space and started the deposition of the loose extracellular matrix. Endothelial cells grow into a wound simultaneously with fibroblasts and undergo angiogenesis. Finally, tissue remodeling takes place to reconstruct the basement membrane by the differentiation of keratinocytes as well as the formation of follicle cells. [43, 49, 50] A scar is an essential part of this natural healing process following any type of damage to the skin. This can occur after a surgical incision or the healing of a wound. As your body makes an effort to close an open wound and protect itself from infection, it replaces injured skin tissue with rapidly generated scar tissue. Scarring is slight when the damaged outer layer of skin is healed by rebuilt tissue. When we damage the thick layer of tissue beneath the skin, rebuilding is more complicated. Our bodies lay down collagen fibers (a protein which is naturally produced by the body) and this usually results in a highly obvious scar. A permanent reminder of the injury is left behind. So, a dressing that can induce scarless healing is needed. Historically, a dressing was usually a piece of material, sometimes cloth, but the use of cobwebs, dung, leaves and honey has also been described. However, modern dressings include gauzes, semipermeable films, low adherent dressings, gels, foams, hydrocolloids, alginates, hydrogels and polysaccharide pastes. Wound dressings are passive, active or interactive. Passive dressings simply provide cover while active or interactive dressings are capable of modifying the physiology of the wound environment. Interactive dressings include hydrocolloids, hydrogels, alginates, foam dressings and antimicrobial dressings. [17, 20, 85] Traditionally dry wound dressings are considered to be good for healing wounds i.e. the wound should be covered with gauze or left open. But it has been observed by Winter [191] that when wound is left open to air without any dressing, a scab i.e. a dry covering covers the wound and decreases the rate of epithelialization. On the other hand, if moist dressing is used in place of dry dressings scab will not form and rate of healing increases as moist dressings provide low oxygen tension which helps in wound healing, these dressings not only keep cells viable which enables them to release growth factors while maintaining contact between them and the healing tissues, but may also modulate or stimulate their proliferation, these dressings decrease the pain at rest, during ambulation and during dressing changes also moist environment allows rapid and efficient delivery of any added antimicrobial agent thus prevent the wound from infection. So, the dressings that create and maintain a mois t environment, however, are now considered to provide the optimal conditions for wound healing. 2 Requirements of an ideal wound care system These characteristics should be present in the ideal wound care system (a) it should be capable of maintaining a high humidity at the wound site, (b) it should be non-toxic, (c) non-allergenic, (d) it can be removed without causing trauma to the wound, (e) it should pe impermeable to bacteria, (f) Thermally insulating, (g) it should be soft to touch, (h) it should allow proper gaseous exchange, (i) it should be free from particulate and toxic product, (j) promote tissue reconstruction processes and (k) it should be cost effective. [20, 165, 177] Out of all the dressings hydrocolloids, alginates and hydrogels each one has its own advantages and limitations but hydrogels are best and have all the characteristics that are needed in an ideal wound dressing. All the above mentioned characteristics can be achieved in hydrogel wound dressings. 3 Hydrogels for Wound Healing Applications Hydrogels are natural or synthetic cross-linked polymers used in a variety of medical and biomedical applications. Hydrogels consist of a matrix of insoluble polymers with up to 96% water content enabling them to donate water molecules to the wound surface and to maintain a moist environment at the wound bed. They are used in the construction of contact lenses, drug-delivery vehicles, wound dressings and as physiological electrodes or sensors. [17] Examples of hydrogel include Aquaform, Intrasite, GranuGel, Nu-Gel, Purilon, Sterigel. These also have the ability to absorb a degree of wound exudate. They transmit moisture vapour and oxygen, but their bacterial and fluid permeability is dependent on the type of secondary dressing used. [20] Hydrogels swell or shrink in aqueous solutions due to the association, dissociation and binding of various ions to polymer chains. These systems may swell in water until an equilibrium state is reached and retain their original shape. The interactions responsible for water sorption by hydrogels include the process of hydration, which is connected to the presence of such chemical groups as -OH, -COOH, -CONH2, -CONH-, and -SO3H and the existence of capillary areas and differences in osmotic pressure. The forces that make hydrogel dissolution impossible are presence of covalent bonds between individual polymer chains, hydrophobic and electrostatic interactions. [46] These are hydrophilic polymer networks which may absorb from 10-20% (an arbitrary lower limit) up to thousands of times their dry weight in water. These may be chemically stable or they may degrade and dissolve. They are called ‘reversible, or ‘physical gels when the networks are held together by molecular entanglements, and/or secondary forces including ionic, H-bonding or hydrophobic forces. [13, 14, 15] Hydrogels are called ‘permanent or ‘chemicalgels when they are covalently-crosslinked networks as shown in 1. Hydrogels can be made by irradiation, freeze-thawing or chemical methods. Out of all the methods, irradiation is considered as a suitable tool for the formation of hydrogels as in this method there is easy control of processing, no need of adding initiators or cross-linkers which are harmful, and have the possibility of formation and sterilization in one step. But as everything has its own advantages and disadvantages this method also has a disadvantage which is hydrogels formed by this method have poor mechanical strength. Nowadays, Freeze thawing technique is generally used to prepare hydrogels having good strength, stability and no crosslinkers and initiators. But the main disadvantage is that the prepared hydrogels have opaque appearance and limited swelling and thermal stability. [120] In comparison to the traditional gauze therapy the application of a hydrogel seems to significantly stimulate wound healing. [11] Various natural and synthetic polymers having good biocompatibility are used to develop hydrogel wound dressing. These polymers include natural polymers such as alginate, chitosan, gelatin and collagen and synthetic polymers such as polyurethane, poly(ethylene glycol), polycaprolactone, poly vinyl pyrrolidone, poly(lactide-co-glycolide), polyacrylonitrile , poly(amino acid), etc. Table 1 below shows different hydrophilic polymers used to synthesize hydrogel matrices. Table 1 Hydrophilic polymers used to synthesize hydrogel matrices. [13] Hydrogels may be classified as homopolymer hydrogels, copolymer hydrogels, multipolymer hydrogels, and interpenetrating polymeric hydrogels. Homopolymer hydrogels are crosslinked networks of one type of hydrophilic monomer unit, whereas copolymer hydrogels are produced by the crosslinking of two comonomer units, one of which must be hydrophilic. Multipolymer hydrogels are produced by the crosslinking of more than three monomers. Finally, interpenetrating polymeric hydrogels are produced by the swelling of a first network in a monomer and the reaction of the latter to form a second intermeshing network structure. [46, 47] Also, it has been shown that the blending of a natural polymer with a synthetic polymer seems to be a good method for obtaining materials having required mechanical and thermal properties in comparison to pure components. It is also a simple method by which suitable shapes such as films, sponges and hydrogels can be obtained easily to realize a variety of biomedical devices. 2 shows healing is faster with the hydrogel dressing than with the gauze dressing. Wound area covered by hydrogel decreases faster with increasing healing period. On the contrary, the wound covered by gauze dressing reduces by only half a percent even after 14 days. [192] 3.1 Natural Hydrogels Natural polymers, such as chitin, chitosan, alginate, collagen, elastin, genipin, gelatin, cellulose etc. have been used for dressing wounds because they play an important role in the healing process. [47] Chitosan is a partially deacetylated form of chitin. Chitin as BeschitinÃ’, Unitika, is also commercially available as dressing in Japan. [28] But as far as chitosan is concerned it is biocompatible, biodegradable, haemostatic, fungistatic [54] and non-toxic and can be successfully used as gels, films, fibres etc. This polymer also show antibacterial properties and possess good wound healing properties. [60, 61, 62] It has many applications as wound dressing, drug delivery device and as scaffold for tissue engineering. [63, 64] Some of the examples of wound dressings are given below which use chitosan as one of the biomaterial. Asymmetric chitosan membranes have been developed by using immersion-precipitation phase-inversion method. [123, 124, 125] These asymmetric chitosan membranes are homogeneous and have porous structure. This membrane was prepared by preheating casted chitosan solution in oven for different time periods for dry phase separation and then immersed in to coagulant tank for wet phase separation and were subsequently freeze-dried. The skin layer acts as the rate controlling barrier for the release of drug and the porous layer provide mechanical support to the skin layer. The water vapor transmission rate, gas permeability, PBS solution absorption, in-vitro degradation, cell culture, bacterial penetration and wound healing test of this dressing were carried out. These membranes are effective in controlling evaporative water loss, showed excellent oxygen permeability and also antibacterial in nature. These are also found to be an urgent hemostat. In another study, silver sulphadiazine was inc orporated as an antimicrobial agent to this asymmetric dressing. The release behaviour of both silver and sulphadiazine ions were studied and found to be significantly different from one another. Silver ions displayed a slow release behavior while sulphadiazine ions exhibited burst effect on first day of the drug release and then slowly tapered off. It is because of the interaction of silver with amino group of chitosan leading to its slow release throughout whereas, as the sulphadiazine ions were free to diffuse through the membrane to reach the wound site thus they showed a burst release. The membranes were further found effective against P. aerugniosa and S. aureus. In one of the papers, novel wound dressings were formed that composed of chitosan film and Minocycline Hydrochloride (MH) and commercial polyurethane film (Tegaderm) as a backing. It is also a useful formulation for the treatment of severe burn wounds. Water vapor and oxygen can permeate the Tegaderm film but water cannot. The tegaderm film support the polymer membrane. [126] In one of the studies, a silver nanocrystalline chitosan (SNC) wound dressing composed of nano-silver and chitosan was constructed by self-assembly and nanotechnology and used for treating deep partial-thickness wounds. In this, sterility and pyrogen testing were performed to ensure biosafety. These dressings promote wound healing and combat infection, and also decrease the risk of silver absorption in comparison with silver sulphadiazine (SSD) dressings. [127] There is also one more method of forming wound dressing composed of chitosan i.e. the formation of polyelectrolyte complex of gum kondagogu (GKG) and chitosan. This complex is formed by the electrostatic interaction between carboxyl group of gum and amine group of chitosan. This method is more advantageous as it avoids the use of organic solvents, chemical crosslinking agents and thus reduces the toxicity and undesirable side effects. In this, diclofenac sodium is used as model drug. The diclofenac loaded complex of gum kondagogu/ chitosan shows drug release which changes with change in pH. The drug release was higher at pH 6.8 as compared to pH 1.2, due to higher swelling of complex at higher pH. This holds a great potential as a natural polymer based delivery device for controlled delivery of drugs like diclofenac sodium for two reasons: (i) to reduce dosing frequency and (ii) lower the gastric toxicity. [152] Semi-interpenetrating polymer networks (SIPNs) composed of chitosan (CS) and poloxamer were prepared in order to improve the mechanical strength of CS. The WVTR was found to be 2508.2 ±65.7 gm−2 day−1, i.e. these can maintain a moist environment at wound site which enhance epithelial cell migration. Also, the in vitro assessment of SIPNs showed proper biodegradation and low cytotoxicity and in vivo is carried out on experimental full thickness wounds in a mouse model and found that the wounds covered with these were completely filled with new epithelium without any significant adverse reactions after 3 weeks. In one of the papers, a kind of surgical wound dressing, the chitosan-gelatin sponge wound dressing (CGSWD) having good antibacterial property is prepared. The in vitro test showed that the antibacterial effect of CGSWD on E. coli K88 is better than that of penicillin, and the effect on S. aureus is also better than that of cefradine. [137] One more wound dressing consists of two separate layers were prepared in which the upper layer is a swellable hydrogel material which can absorb exudates and also serve as mechanical and microbial barrier while lower layer is a chitosan acetate foam incorporated with the anti-microbial agent chlorherxidine gluconate. [183] The antimicrobial activity is checked by the Bauer-Kirby Disk Diffusion Test, inhibition zones can be clearly seen around the discs of chitosan acetate foams incorporated with chlorhexidine gluconate, in culture plates inoculated with either Gram-negative or positive bacteria showing that the dressing is antimicrobial in nature. Blending is a convenient and effective method to improve physical and mechanical properties of hydrogels. So modification of chitosan is done by blending with other polymers like cellulose. [178] In this, E. coli and S. aureus were used as the test bacteria to examine the antibacterial properties of chitosan, cellulose and chitosan/cellulose blends. The numbers of colony of these bacteria formed on the test membranes are shown in s 5 and 6. It was noted that the numbers of colony of all test bacteria formed on the chitosan/cellulose blend membranes were decreased with the increase of chitosan concentration. These blends are more effective against E. coli than that of S. aureus, as indicated by the lower colony unit. Thus these dressings are suitable to use as an antimicrobial wound dressing. Chitosan due to its structural properties has the ability to heal wounds without scar formation. [198] Since chitosan is composed of D-glucosamine, which is also the component present in the disaccharide subunits of hyaluronic acid, chitosan tries to structurally mimic hyaluronic acid and exerts similar effects. [197] It has been known that the fetal wound healing takes place without fibrosis or scar formation due to the presence of hyaluronic acid. [199] In one of the studies, Chitosan as a semi-permeable biological dressing maintains a moist environment and prevent the wound site from dehydration and contamination. In addition, digital colour separation analysis of donor site scars demonstrated an earlier return to normal skin colour at chitosan-treated areas as shown in 7. [196] Collagen is also a biopolymer that is used as a polymer for making wound dressing and drug delivery devices as it is biocompatible and biocompatibility of a material applied to wound tissue is a prerequisite for optimal wound environment and facilitation of the healing processes. A new collagen dressing with gentamycin or amikacin was prepared in one of the research work and these follow the basic requirement of antibacterial bandages. The dressing is composed of two collagen biomaterials—membrane and sponge—both possessing good tissue biocompatibility. These dressing released antibiotics slowly and show the antibacterial treatment in experimentally infected superficial wounds in mice. Thus, it can be used for the treatment of infected wounds in humans. [26] As discussed previously that both chitosan and collagen are excellent materials that can be used as wound dressing materials. So it has been seen that if both are used simultaneously then what will be the effect. It is found that the wound dressings composed of chitosan crosslinked collagen sponge (CCCS) enhance the diabetic wound healing. Collagen crosslinked with chitosan showed several advantages required for wound dressing, including the uniform and porous ultrastructure, less water imbibition, small interval porosity, and high resistance to collagenase digestion and slow release of FGF from CCCS/FGF. [138] Following moist healing concept, alginates which are able to absorb exudates from wound have become one of the most important materials for wound management. [52, 53, 54, 55, 56] In this particular field, the properties of alginate fibers are unparalleled in many respects. Alginate based products form a gel and effective in removing out of the wound on the contrary to traditional cotton and viscose fibres, which can entrap in the wound developing discomfort during dressing removal. [55] Also, the alginate fibres are non-toxic, non-carcinogenic, non-allergic, haemostatic, biocompatible, of reasonable strength, capable of being sterilized and easily processable. Sorbsanâ„ ¢ was first commercialized in 1981 and after that there were many dressings launched. The alginate fibers can be converted into wound dressings by using a number of textile processes. Because of its simplicity and also the high absorbency of the product, nonwoven is the main form of alginate wound dressings. [19] The antimicrobial action of alginate dressing can be seen as in 8 which shows the antimicrobial action of silver containing alginate fibers against E. Coli. There was 100% reduction in bacteria count within 5 hr after the fibers were placed in contact with solutions containing the bacteria. Sorbsan alginate fibers showed some antimicrobial activity while AquacelTM (made of carboxymethyl cellulose), does not show any antimicrobial effect. [18] Gelatin widely found in nature and is the major constituent of skin, bones, and connective tissue. Gelatin can easily be obtained by a controlled hydrolysis of the fibrous insoluble protein, collagen. [195] This is also used in number of biomedical applications like wound dressings. Hydrogel wound dressing from gelatin, oxidized alginate and borax were prepared and the composite matrix promotes wound healing because of alginate, has haemostatic effect of gelatin and is antiseptic because of borax. The water vapour transmission rate (WVTR) of the hydrogel was calculated and found to be 2686 ±124 g/m2/day indicating that this hydrogel can maintain a proper fluid balance at the wound site which helps in cell migration. 2 shows the loss of water vapour with time through the hydrogel when placed in a moisture rich environment. [119] Genipin has been used to crosslink chitosan membranes to control swelling ratio and mechanical properties. It increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. It had significantly less cytotoxicity for human fibroblasts and slower degradation rate compared to the glutaraldehyde-crosslinked membrane. This genipin crosslinked chitosan membrane can be successfully used as a wound dressing. [132] Bacterial cellulose is a natural polymer consisting of microfibrils containing glucan chains bound together by hydrogen bonds. Bacterial cellulose with chitosan combines properties such as bioactivity, biocompatibility, and biodegradability of the two biopolymers and form an ideal material for dressing wounds. [66, 67] These are antibacterial and scar preventive in nature too. Since natural polymers have been considered limited in their applications for wound-dressing materials as their low mechanical properties and shortage of processing. So we move towards the synthetic polymers that can be used for wound healing applications. 3.2 Synthetic Hydrogels Synthetic polymers are also being used successfully in biomedical applications as one of the materials because of their wide range of mechanical properties, suitability for easily forming into a variety of different shapes, and low production costs. In an ideal dressing both the characteristics i.e. antimicrobial ability and moist healing environment should be present, so in order to prevent the wound from dehydration and bacterial penetration a new dressing composed of polyurethane is designed in such a way that the upper layer of the dressing is microporous (pore size Polyvinyl pyrrolidone (PVP) is one of the most widely used synthetic polymers in medicine because of its solubility in water and its extremely low cytotoxicity. Hydrogels prepared by radiation crosslinking of an aqueous solution of polyvinyl pyrrolidone (PVP) have been used as wound dressing. [21] These are biocompatible, reduces pain, easily replacable, permeable to oxygen, maintain moist environment at the wound site. Polyvinyl alcohol (PVA) is a well-known polymer because it is biocompatible and have required mechanical properties and polyethylene oxide (PEO) is a hydrophilic semicrystalline polyether which is biocompatible, non toxic, non polar, non antigenic and non immunogenic and is highly desirable in most biomedical applications requiring contact with physiological fluids. A hydrogel composed of PEO for wound dressing is prepared and PVA is added to give toughness to the hydrogel by electron beam irradiation and found that these hydrogels showed satisfactory properties for wound dressing that has been evaluated by creating wound on the back of the marmots. [157] The hydrogel gives a wet environment to wounds which causes faster healing compared with the gauze dressing with a dry environment. The weight of the hydrogel increases quickly at the earlier stages, up to 4 days, due to absorption of effusion produced from the wound as shown in Table 2. After that, the production of effusion from the wound ceases and the weight of the hydrogel decreases due to evaporation of the water in the hydrogel. This means that the healing of wound proceeds smoothly with time. The hydrogel can be peeled off easily from the wound at the time of removal. Table 2 Absorption of effusion from wound of dressing during healing. [157] The toughness of PEO hydrogel is improved by the addition of PVA and tensile strength is measured and found that as shown in 10 and 11, the tensile strength and elongation decrease with increasing dose because of the increase of crosslinking. Various synthetic polymers as discussed above are used for wound dressing applications. But the major problem with these materials is their biocompatibility characteristics are often unsatisfactory and their interaction with living tissues is a major problem. So a combination of both natural and synthetic polymers is the better option to make a hydrogel having biocompatibility and desired mechanical strength. 3.3 Blended hydrogels Since both the natural and synthetic polymers have their own advantages and disadvantages so a combination of natural and synthetic polymers can endow the optimal properties necessary for wound repair. [47] The combination of natural and synthetic polymers is used in the biomedical, bioengineering and biotechnology field nowadays because of their great potential. A blended hydrogel composed of polyvinyl alcohol/polyvinyl pyrrolidone and charcoal were prepared by single ‘‘freezing and thawing or two-step ‘‘freezing and thawing and ÃŽ ³-ray irradiation and applied as wound dressing. It is found that the absorption of S. aureus and P. aeruginosa by charcoal/PVA/PVP hydrogels was larger than those by PVA/PVP hydrogels, this is due to the absorption and attachment capability of bacteria by charcoal, this can be shown in 12 given below. [22] The most classical way of fabricating a CS based wound dressing has been to design an asymmetric composite structure. In this method, the Cotton fabric was coated with chitosan (CS) and polyethylene glycol (PEG) followed by freeze-drying. The outer dense layer helps in preventing the microbial passage across the dressing and provides a rate controlling barrier for water vapor permeation, while the inner porous layer provides a high surface area for the exudates absorption. For the absorption of wound exudates porosity is the prime requirement in a dressing. It has been found that these dressings have the porosity 54-70% and the pore size was in the range of 75-120 µm. [45] The increase in the PEG content in the blend composition led to an enhanced destabilization of pores, leading to an increase in the pore size with elongated morphology. There seems to be phase separation between the two components which is an important factor for the observed behavior of the porous structure. Cot ton fabric has been used as the support layer for the CS-PEG layer and leads to very thin and light weight structures. The structure of the dressing has been designed in such a way that it leads to the high porosity of the bulk structure. The thickness of CS coating plays an important role in the development of the porosity on the surface. The influence of the CS thickness on the surface morphology is presented in 13 given below. PEG addition to CS makes significant alteration in the surface morphology of this CS-PEG/cotton membrane (freeze-dried), henceforth known as CPC membrane. There is a distinct trend in the loss of inherent elongated porous structure in membranes and formation of the partially collapsed porosity takes place due to the PEG addition. This suggests that a very limited interaction between CS and PEG exists which is reflected in the observed surface morphology. It has been observed that higher the amount of PEG, the higher is the pore destabilization leading to larger pores. This is evident from the morphology of the CPC membrane at 50% PEG-20 content as shown in 14.45 On the above matrix, the addition of PVP and drug followed by coating on the cotton fabric and freeze drying of the coated matrix is also done. It has been found that the drug Hydrogels for Wound Healing Applications Hydrogels for Wound Healing Applications Abstract This chapter discusses about the hydrogels both natural and synthetic that can be used for wound healing applications. Also it discusses briefly about the various novel techniques that have been developed recently. Keywords: Hydrogels; Chitosan, Antimicrobial; Grafting; Blending; Wound dressing; Wound healing; Gene therapy, Stem cell therapy, Skin Engineering, pH and Thermosensitive polymers. 1 Introduction Healthcare is an essential aspect of human survival. So many biopolymers have generated interest in a number of biomedical applications. Wound management is one such area where management of cuts, ulcers, and sores needs dressings which help in promoting rapid wound healing in order to obtain both functional and cosmetic results. [45] There are different kinds of wound management products: staples or sutures, dressings or bandages, surgical sealants and adhesives, skin substitutes, and other biomaterials. [51] Human skin provides an effective barrier to microbial penetration and subsequent infection. However, once the wound has been developed in this barrier, the infection chances increases. In case of chronic wounds, the colonization and infection potential increases as the result of the presence of avascular eschar which provides an environment for the uninhibited growth of microorganisms. [89] The rate of infection is related to the type of wound, general wound care, and local health of the patient. [88, 90] For avoiding infection, good clinical practices are needed. The management of chronic wounds is a very costly practice and it also places an enormous drain on healthcare resources; studies have calculated the cost of wounds to the NHS to be about  £1bn a year. [166] So for lowering this cost such wound management products are needed that are more economical and effective. Out of all the above wound management products, here in this chapter we will discuss more about the wound dressings that will provide an optimal healing environment to the wound. A dressing is an adjunct used by a person for application to a wound in order to promote healing and/or prevent further harm. It is designed to be in direct contact with the wound, so it is different from the bandage in the manner that bandages are normally used to hold dressing in place. A wound is a break in the epithelial integrity of the skin and may be accompanied by disruption of the structure and function of underlying normal tissue. Wounds can be divided into four categories based on their appearance and stage of healing: Necrotic, sloughing, granulating and epithelializing wounds. [87] Wounds cause discomfort and are more prone to infection and other troublesome complications. [75] Some diseases like diabetes, ischaemia and conditions like malnourishment, ageing, local infection, local tissue damage due to burn leads to delay in wound healing. Infection is a major complication of burn injury and is responsible for 50-75% of hospital deaths. [76] Human skin has one of the greatest capacities to regenerate itself amongst all of the tissues in our body. It constantly replaces old cells with new cells, enabling it to repair itself when damaged. Wound healing is a complex-physiologic process, which consists of three overlapping phases: inflammatory, proliferative and remodeling phases. The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors. During the inflammation process, neutrophils are the first leukocytes which come at the site of injury to rid it from bacterial contamination. Then, the monocytes and their conversion to macrophages initiate tissue repair by releasing a number of biologically active substances and growth factors that are necessary for the initi ation of tissue formation process. In the third process, fibroblasts proliferate and migrate into the wound space and started the deposition of the loose extracellular matrix. Endothelial cells grow into a wound simultaneously with fibroblasts and undergo angiogenesis. Finally, tissue remodeling takes place to reconstruct the basement membrane by the differentiation of keratinocytes as well as the formation of follicle cells. [43, 49, 50] A scar is an essential part of this natural healing process following any type of damage to the skin. This can occur after a surgical incision or the healing of a wound. As your body makes an effort to close an open wound and protect itself from infection, it replaces injured skin tissue with rapidly generated scar tissue. Scarring is slight when the damaged outer layer of skin is healed by rebuilt tissue. When we damage the thick layer of tissue beneath the skin, rebuilding is more complicated. Our bodies lay down collagen fibers (a protein which is naturally produced by the body) and this usually results in a highly obvious scar. A permanent reminder of the injury is left behind. So, a dressing that can induce scarless healing is needed. Historically, a dressing was usually a piece of material, sometimes cloth, but the use of cobwebs, dung, leaves and honey has also been described. However, modern dressings include gauzes, semipermeable films, low adherent dressings, gels, foams, hydrocolloids, alginates, hydrogels and polysaccharide pastes. Wound dressings are passive, active or interactive. Passive dressings simply provide cover while active or interactive dressings are capable of modifying the physiology of the wound environment. Interactive dressings include hydrocolloids, hydrogels, alginates, foam dressings and antimicrobial dressings. [17, 20, 85] Traditionally dry wound dressings are considered to be good for healing wounds i.e. the wound should be covered with gauze or left open. But it has been observed by Winter [191] that when wound is left open to air without any dressing, a scab i.e. a dry covering covers the wound and decreases the rate of epithelialization. On the other hand, if moist dressing is used in place of dry dressings scab will not form and rate of healing increases as moist dressings provide low oxygen tension which helps in wound healing, these dressings not only keep cells viable which enables them to release growth factors while maintaining contact between them and the healing tissues, but may also modulate or stimulate their proliferation, these dressings decrease the pain at rest, during ambulation and during dressing changes also moist environment allows rapid and efficient delivery of any added antimicrobial agent thus prevent the wound from infection. So, the dressings that create and maintain a mois t environment, however, are now considered to provide the optimal conditions for wound healing. 2 Requirements of an ideal wound care system These characteristics should be present in the ideal wound care system (a) it should be capable of maintaining a high humidity at the wound site, (b) it should be non-toxic, (c) non-allergenic, (d) it can be removed without causing trauma to the wound, (e) it should pe impermeable to bacteria, (f) Thermally insulating, (g) it should be soft to touch, (h) it should allow proper gaseous exchange, (i) it should be free from particulate and toxic product, (j) promote tissue reconstruction processes and (k) it should be cost effective. [20, 165, 177] Out of all the dressings hydrocolloids, alginates and hydrogels each one has its own advantages and limitations but hydrogels are best and have all the characteristics that are needed in an ideal wound dressing. All the above mentioned characteristics can be achieved in hydrogel wound dressings. 3 Hydrogels for Wound Healing Applications Hydrogels are natural or synthetic cross-linked polymers used in a variety of medical and biomedical applications. Hydrogels consist of a matrix of insoluble polymers with up to 96% water content enabling them to donate water molecules to the wound surface and to maintain a moist environment at the wound bed. They are used in the construction of contact lenses, drug-delivery vehicles, wound dressings and as physiological electrodes or sensors. [17] Examples of hydrogel include Aquaform, Intrasite, GranuGel, Nu-Gel, Purilon, Sterigel. These also have the ability to absorb a degree of wound exudate. They transmit moisture vapour and oxygen, but their bacterial and fluid permeability is dependent on the type of secondary dressing used. [20] Hydrogels swell or shrink in aqueous solutions due to the association, dissociation and binding of various ions to polymer chains. These systems may swell in water until an equilibrium state is reached and retain their original shape. The interactions responsible for water sorption by hydrogels include the process of hydration, which is connected to the presence of such chemical groups as -OH, -COOH, -CONH2, -CONH-, and -SO3H and the existence of capillary areas and differences in osmotic pressure. The forces that make hydrogel dissolution impossible are presence of covalent bonds between individual polymer chains, hydrophobic and electrostatic interactions. [46] These are hydrophilic polymer networks which may absorb from 10-20% (an arbitrary lower limit) up to thousands of times their dry weight in water. These may be chemically stable or they may degrade and dissolve. They are called ‘reversible, or ‘physical gels when the networks are held together by molecular entanglements, and/or secondary forces including ionic, H-bonding or hydrophobic forces. [13, 14, 15] Hydrogels are called ‘permanent or ‘chemicalgels when they are covalently-crosslinked networks as shown in 1. Hydrogels can be made by irradiation, freeze-thawing or chemical methods. Out of all the methods, irradiation is considered as a suitable tool for the formation of hydrogels as in this method there is easy control of processing, no need of adding initiators or cross-linkers which are harmful, and have the possibility of formation and sterilization in one step. But as everything has its own advantages and disadvantages this method also has a disadvantage which is hydrogels formed by this method have poor mechanical strength. Nowadays, Freeze thawing technique is generally used to prepare hydrogels having good strength, stability and no crosslinkers and initiators. But the main disadvantage is that the prepared hydrogels have opaque appearance and limited swelling and thermal stability. [120] In comparison to the traditional gauze therapy the application of a hydrogel seems to significantly stimulate wound healing. [11] Various natural and synthetic polymers having good biocompatibility are used to develop hydrogel wound dressing. These polymers include natural polymers such as alginate, chitosan, gelatin and collagen and synthetic polymers such as polyurethane, poly(ethylene glycol), polycaprolactone, poly vinyl pyrrolidone, poly(lactide-co-glycolide), polyacrylonitrile , poly(amino acid), etc. Table 1 below shows different hydrophilic polymers used to synthesize hydrogel matrices. Table 1 Hydrophilic polymers used to synthesize hydrogel matrices. [13] Hydrogels may be classified as homopolymer hydrogels, copolymer hydrogels, multipolymer hydrogels, and interpenetrating polymeric hydrogels. Homopolymer hydrogels are crosslinked networks of one type of hydrophilic monomer unit, whereas copolymer hydrogels are produced by the crosslinking of two comonomer units, one of which must be hydrophilic. Multipolymer hydrogels are produced by the crosslinking of more than three monomers. Finally, interpenetrating polymeric hydrogels are produced by the swelling of a first network in a monomer and the reaction of the latter to form a second intermeshing network structure. [46, 47] Also, it has been shown that the blending of a natural polymer with a synthetic polymer seems to be a good method for obtaining materials having required mechanical and thermal properties in comparison to pure components. It is also a simple method by which suitable shapes such as films, sponges and hydrogels can be obtained easily to realize a variety of biomedical devices. 2 shows healing is faster with the hydrogel dressing than with the gauze dressing. Wound area covered by hydrogel decreases faster with increasing healing period. On the contrary, the wound covered by gauze dressing reduces by only half a percent even after 14 days. [192] 3.1 Natural Hydrogels Natural polymers, such as chitin, chitosan, alginate, collagen, elastin, genipin, gelatin, cellulose etc. have been used for dressing wounds because they play an important role in the healing process. [47] Chitosan is a partially deacetylated form of chitin. Chitin as BeschitinÃ’, Unitika, is also commercially available as dressing in Japan. [28] But as far as chitosan is concerned it is biocompatible, biodegradable, haemostatic, fungistatic [54] and non-toxic and can be successfully used as gels, films, fibres etc. This polymer also show antibacterial properties and possess good wound healing properties. [60, 61, 62] It has many applications as wound dressing, drug delivery device and as scaffold for tissue engineering. [63, 64] Some of the examples of wound dressings are given below which use chitosan as one of the biomaterial. Asymmetric chitosan membranes have been developed by using immersion-precipitation phase-inversion method. [123, 124, 125] These asymmetric chitosan membranes are homogeneous and have porous structure. This membrane was prepared by preheating casted chitosan solution in oven for different time periods for dry phase separation and then immersed in to coagulant tank for wet phase separation and were subsequently freeze-dried. The skin layer acts as the rate controlling barrier for the release of drug and the porous layer provide mechanical support to the skin layer. The water vapor transmission rate, gas permeability, PBS solution absorption, in-vitro degradation, cell culture, bacterial penetration and wound healing test of this dressing were carried out. These membranes are effective in controlling evaporative water loss, showed excellent oxygen permeability and also antibacterial in nature. These are also found to be an urgent hemostat. In another study, silver sulphadiazine was inc orporated as an antimicrobial agent to this asymmetric dressing. The release behaviour of both silver and sulphadiazine ions were studied and found to be significantly different from one another. Silver ions displayed a slow release behavior while sulphadiazine ions exhibited burst effect on first day of the drug release and then slowly tapered off. It is because of the interaction of silver with amino group of chitosan leading to its slow release throughout whereas, as the sulphadiazine ions were free to diffuse through the membrane to reach the wound site thus they showed a burst release. The membranes were further found effective against P. aerugniosa and S. aureus. In one of the papers, novel wound dressings were formed that composed of chitosan film and Minocycline Hydrochloride (MH) and commercial polyurethane film (Tegaderm) as a backing. It is also a useful formulation for the treatment of severe burn wounds. Water vapor and oxygen can permeate the Tegaderm film but water cannot. The tegaderm film support the polymer membrane. [126] In one of the studies, a silver nanocrystalline chitosan (SNC) wound dressing composed of nano-silver and chitosan was constructed by self-assembly and nanotechnology and used for treating deep partial-thickness wounds. In this, sterility and pyrogen testing were performed to ensure biosafety. These dressings promote wound healing and combat infection, and also decrease the risk of silver absorption in comparison with silver sulphadiazine (SSD) dressings. [127] There is also one more method of forming wound dressing composed of chitosan i.e. the formation of polyelectrolyte complex of gum kondagogu (GKG) and chitosan. This complex is formed by the electrostatic interaction between carboxyl group of gum and amine group of chitosan. This method is more advantageous as it avoids the use of organic solvents, chemical crosslinking agents and thus reduces the toxicity and undesirable side effects. In this, diclofenac sodium is used as model drug. The diclofenac loaded complex of gum kondagogu/ chitosan shows drug release which changes with change in pH. The drug release was higher at pH 6.8 as compared to pH 1.2, due to higher swelling of complex at higher pH. This holds a great potential as a natural polymer based delivery device for controlled delivery of drugs like diclofenac sodium for two reasons: (i) to reduce dosing frequency and (ii) lower the gastric toxicity. [152] Semi-interpenetrating polymer networks (SIPNs) composed of chitosan (CS) and poloxamer were prepared in order to improve the mechanical strength of CS. The WVTR was found to be 2508.2 ±65.7 gm−2 day−1, i.e. these can maintain a moist environment at wound site which enhance epithelial cell migration. Also, the in vitro assessment of SIPNs showed proper biodegradation and low cytotoxicity and in vivo is carried out on experimental full thickness wounds in a mouse model and found that the wounds covered with these were completely filled with new epithelium without any significant adverse reactions after 3 weeks. In one of the papers, a kind of surgical wound dressing, the chitosan-gelatin sponge wound dressing (CGSWD) having good antibacterial property is prepared. The in vitro test showed that the antibacterial effect of CGSWD on E. coli K88 is better than that of penicillin, and the effect on S. aureus is also better than that of cefradine. [137] One more wound dressing consists of two separate layers were prepared in which the upper layer is a swellable hydrogel material which can absorb exudates and also serve as mechanical and microbial barrier while lower layer is a chitosan acetate foam incorporated with the anti-microbial agent chlorherxidine gluconate. [183] The antimicrobial activity is checked by the Bauer-Kirby Disk Diffusion Test, inhibition zones can be clearly seen around the discs of chitosan acetate foams incorporated with chlorhexidine gluconate, in culture plates inoculated with either Gram-negative or positive bacteria showing that the dressing is antimicrobial in nature. Blending is a convenient and effective method to improve physical and mechanical properties of hydrogels. So modification of chitosan is done by blending with other polymers like cellulose. [178] In this, E. coli and S. aureus were used as the test bacteria to examine the antibacterial properties of chitosan, cellulose and chitosan/cellulose blends. The numbers of colony of these bacteria formed on the test membranes are shown in s 5 and 6. It was noted that the numbers of colony of all test bacteria formed on the chitosan/cellulose blend membranes were decreased with the increase of chitosan concentration. These blends are more effective against E. coli than that of S. aureus, as indicated by the lower colony unit. Thus these dressings are suitable to use as an antimicrobial wound dressing. Chitosan due to its structural properties has the ability to heal wounds without scar formation. [198] Since chitosan is composed of D-glucosamine, which is also the component present in the disaccharide subunits of hyaluronic acid, chitosan tries to structurally mimic hyaluronic acid and exerts similar effects. [197] It has been known that the fetal wound healing takes place without fibrosis or scar formation due to the presence of hyaluronic acid. [199] In one of the studies, Chitosan as a semi-permeable biological dressing maintains a moist environment and prevent the wound site from dehydration and contamination. In addition, digital colour separation analysis of donor site scars demonstrated an earlier return to normal skin colour at chitosan-treated areas as shown in 7. [196] Collagen is also a biopolymer that is used as a polymer for making wound dressing and drug delivery devices as it is biocompatible and biocompatibility of a material applied to wound tissue is a prerequisite for optimal wound environment and facilitation of the healing processes. A new collagen dressing with gentamycin or amikacin was prepared in one of the research work and these follow the basic requirement of antibacterial bandages. The dressing is composed of two collagen biomaterials—membrane and sponge—both possessing good tissue biocompatibility. These dressing released antibiotics slowly and show the antibacterial treatment in experimentally infected superficial wounds in mice. Thus, it can be used for the treatment of infected wounds in humans. [26] As discussed previously that both chitosan and collagen are excellent materials that can be used as wound dressing materials. So it has been seen that if both are used simultaneously then what will be the effect. It is found that the wound dressings composed of chitosan crosslinked collagen sponge (CCCS) enhance the diabetic wound healing. Collagen crosslinked with chitosan showed several advantages required for wound dressing, including the uniform and porous ultrastructure, less water imbibition, small interval porosity, and high resistance to collagenase digestion and slow release of FGF from CCCS/FGF. [138] Following moist healing concept, alginates which are able to absorb exudates from wound have become one of the most important materials for wound management. [52, 53, 54, 55, 56] In this particular field, the properties of alginate fibers are unparalleled in many respects. Alginate based products form a gel and effective in removing out of the wound on the contrary to traditional cotton and viscose fibres, which can entrap in the wound developing discomfort during dressing removal. [55] Also, the alginate fibres are non-toxic, non-carcinogenic, non-allergic, haemostatic, biocompatible, of reasonable strength, capable of being sterilized and easily processable. Sorbsanâ„ ¢ was first commercialized in 1981 and after that there were many dressings launched. The alginate fibers can be converted into wound dressings by using a number of textile processes. Because of its simplicity and also the high absorbency of the product, nonwoven is the main form of alginate wound dressings. [19] The antimicrobial action of alginate dressing can be seen as in 8 which shows the antimicrobial action of silver containing alginate fibers against E. Coli. There was 100% reduction in bacteria count within 5 hr after the fibers were placed in contact with solutions containing the bacteria. Sorbsan alginate fibers showed some antimicrobial activity while AquacelTM (made of carboxymethyl cellulose), does not show any antimicrobial effect. [18] Gelatin widely found in nature and is the major constituent of skin, bones, and connective tissue. Gelatin can easily be obtained by a controlled hydrolysis of the fibrous insoluble protein, collagen. [195] This is also used in number of biomedical applications like wound dressings. Hydrogel wound dressing from gelatin, oxidized alginate and borax were prepared and the composite matrix promotes wound healing because of alginate, has haemostatic effect of gelatin and is antiseptic because of borax. The water vapour transmission rate (WVTR) of the hydrogel was calculated and found to be 2686 ±124 g/m2/day indicating that this hydrogel can maintain a proper fluid balance at the wound site which helps in cell migration. 2 shows the loss of water vapour with time through the hydrogel when placed in a moisture rich environment. [119] Genipin has been used to crosslink chitosan membranes to control swelling ratio and mechanical properties. It increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. It had significantly less cytotoxicity for human fibroblasts and slower degradation rate compared to the glutaraldehyde-crosslinked membrane. This genipin crosslinked chitosan membrane can be successfully used as a wound dressing. [132] Bacterial cellulose is a natural polymer consisting of microfibrils containing glucan chains bound together by hydrogen bonds. Bacterial cellulose with chitosan combines properties such as bioactivity, biocompatibility, and biodegradability of the two biopolymers and form an ideal material for dressing wounds. [66, 67] These are antibacterial and scar preventive in nature too. Since natural polymers have been considered limited in their applications for wound-dressing materials as their low mechanical properties and shortage of processing. So we move towards the synthetic polymers that can be used for wound healing applications. 3.2 Synthetic Hydrogels Synthetic polymers are also being used successfully in biomedical applications as one of the materials because of their wide range of mechanical properties, suitability for easily forming into a variety of different shapes, and low production costs. In an ideal dressing both the characteristics i.e. antimicrobial ability and moist healing environment should be present, so in order to prevent the wound from dehydration and bacterial penetration a new dressing composed of polyurethane is designed in such a way that the upper layer of the dressing is microporous (pore size Polyvinyl pyrrolidone (PVP) is one of the most widely used synthetic polymers in medicine because of its solubility in water and its extremely low cytotoxicity. Hydrogels prepared by radiation crosslinking of an aqueous solution of polyvinyl pyrrolidone (PVP) have been used as wound dressing. [21] These are biocompatible, reduces pain, easily replacable, permeable to oxygen, maintain moist environment at the wound site. Polyvinyl alcohol (PVA) is a well-known polymer because it is biocompatible and have required mechanical properties and polyethylene oxide (PEO) is a hydrophilic semicrystalline polyether which is biocompatible, non toxic, non polar, non antigenic and non immunogenic and is highly desirable in most biomedical applications requiring contact with physiological fluids. A hydrogel composed of PEO for wound dressing is prepared and PVA is added to give toughness to the hydrogel by electron beam irradiation and found that these hydrogels showed satisfactory properties for wound dressing that has been evaluated by creating wound on the back of the marmots. [157] The hydrogel gives a wet environment to wounds which causes faster healing compared with the gauze dressing with a dry environment. The weight of the hydrogel increases quickly at the earlier stages, up to 4 days, due to absorption of effusion produced from the wound as shown in Table 2. After that, the production of effusion from the wound ceases and the weight of the hydrogel decreases due to evaporation of the water in the hydrogel. This means that the healing of wound proceeds smoothly with time. The hydrogel can be peeled off easily from the wound at the time of removal. Table 2 Absorption of effusion from wound of dressing during healing. [157] The toughness of PEO hydrogel is improved by the addition of PVA and tensile strength is measured and found that as shown in 10 and 11, the tensile strength and elongation decrease with increasing dose because of the increase of crosslinking. Various synthetic polymers as discussed above are used for wound dressing applications. But the major problem with these materials is their biocompatibility characteristics are often unsatisfactory and their interaction with living tissues is a major problem. So a combination of both natural and synthetic polymers is the better option to make a hydrogel having biocompatibility and desired mechanical strength. 3.3 Blended hydrogels Since both the natural and synthetic polymers have their own advantages and disadvantages so a combination of natural and synthetic polymers can endow the optimal properties necessary for wound repair. [47] The combination of natural and synthetic polymers is used in the biomedical, bioengineering and biotechnology field nowadays because of their great potential. A blended hydrogel composed of polyvinyl alcohol/polyvinyl pyrrolidone and charcoal were prepared by single ‘‘freezing and thawing or two-step ‘‘freezing and thawing and ÃŽ ³-ray irradiation and applied as wound dressing. It is found that the absorption of S. aureus and P. aeruginosa by charcoal/PVA/PVP hydrogels was larger than those by PVA/PVP hydrogels, this is due to the absorption and attachment capability of bacteria by charcoal, this can be shown in 12 given below. [22] The most classical way of fabricating a CS based wound dressing has been to design an asymmetric composite structure. In this method, the Cotton fabric was coated with chitosan (CS) and polyethylene glycol (PEG) followed by freeze-drying. The outer dense layer helps in preventing the microbial passage across the dressing and provides a rate controlling barrier for water vapor permeation, while the inner porous layer provides a high surface area for the exudates absorption. For the absorption of wound exudates porosity is the prime requirement in a dressing. It has been found that these dressings have the porosity 54-70% and the pore size was in the range of 75-120 µm. [45] The increase in the PEG content in the blend composition led to an enhanced destabilization of pores, leading to an increase in the pore size with elongated morphology. There seems to be phase separation between the two components which is an important factor for the observed behavior of the porous structure. Cot ton fabric has been used as the support layer for the CS-PEG layer and leads to very thin and light weight structures. The structure of the dressing has been designed in such a way that it leads to the high porosity of the bulk structure. The thickness of CS coating plays an important role in the development of the porosity on the surface. The influence of the CS thickness on the surface morphology is presented in 13 given below. PEG addition to CS makes significant alteration in the surface morphology of this CS-PEG/cotton membrane (freeze-dried), henceforth known as CPC membrane. There is a distinct trend in the loss of inherent elongated porous structure in membranes and formation of the partially collapsed porosity takes place due to the PEG addition. This suggests that a very limited interaction between CS and PEG exists which is reflected in the observed surface morphology. It has been observed that higher the amount of PEG, the higher is the pore destabilization leading to larger pores. This is evident from the morphology of the CPC membrane at 50% PEG-20 content as shown in 14.45 On the above matrix, the addition of PVP and drug followed by coating on the cotton fabric and freeze drying of the coated matrix is also done. It has been found that the drug