8.3.2 Honey in Alginate Wound Dressings
MEDIHONEY® antimicrobial honey dressings (Derma Sciences) are a new and unique line of dressings. Active Manuka Honey (Leptospermum scoparium) has been reported to have powerful and long-lasting antimicrobial activity similar to that of ionic silver; however, there seems to be no associated toxicity as found with ionic silver. This dressing has been reported to significantly improve the rate of healing and reduce the MRSA burden, and effectively assist in debridement. It also helps to manage the moisture balance and control the odour often associated with infected or highly colonised wounds. Honey gelled with calcium alginate forms a rubbery sheet similar to hydrocolloids and forms a softer gel upon absorbing exudates, which keeps honey in contact with the wound [35]. MEDIHONEY® gel sheets and ApinateTM dressings (Comvita, honey-impregnated mechanically bonded calcium alginate fibre) and Algivon® dressings (Advancis) create a barrier against antibiotic-resistant strains and other wound pathogens, which reduces the risk of infection. The outflow of exudates, induced by osmotic action, removes the wound bacteria, endotoxins debris and slough. As an optimal healing environment enhances granulation and epithelialization, these products are suitable for the treatment of leg ulcers, burns, donor graft sites and infected wounds.
a turning point, where one needs a very critical look at its potential to live up to expectations [36, 37]. There have been various attempts to develop wound-healing devices using chitosan due to its inherent wound-healing potential. Both chitin and chitosan possess many properties that are advantageous for wound healing such as biocompatibility, biodegradability, haemostatic activity, healing acceleration, nontoxicity, and adsorption and anti-infection properties. Chitosan provides a nonprotein matrix for 3D tissue growth and activates macrophages for tumouricidal activity; it also stimulates cell proliferation and histoarchitectural tissue organisation [38]. Chitosan is a haemostat and helps in natural blood clotting and blocks nerve endings which reduces pain (Figure 8.5) [39]. Chitosan will gradually depolymerise to release N-acetyl-β-D-glucosamine, which initiates fibroblast proliferation and helps in ordered collagen deposition, and stimulates an increased level of natural hyaluronic acid synthesis at the wound site, it also helps in faster wound healing and scar prevention.
Minimises scarring
Strengthens new tissue
Provides protein for healing
Absorbs fluids from inflammation Blocks nerve
endings to reduce pain Encourages
natural blood clotting
Forms barrier against infection
Provides scaffold for cell growth
Figure 8.5 Schematic representation of the benefits of chitosan in wound healing.
Reproduced with permission from W. Paul, R. Deepa, T.V. Anilkumar and C.P. Sharma in Chitin and Chitosan Derivatives: Advances in Drug Discovery and
Developments, Ed., S-K. Kim, CRC Press/Taylor & Francis Group, London, UK, 2013, p.243. ©2013, CRC Press [39]
Various dressings for wound healing, based on chitin and chitosan, have been developed and are on the market. These are available in the form of nonwovens, nanofibrils, composites, films and sponges. The most prominent among them is the HemCon® haemostatic bandage [US Food and Drug Administration (FDA) approved in 2002], which was extensively used and claimed to have saved the lives of hundreds of US soldiers serving in Iraq and Afghanistan. However, because of mixed results, these haemostatic bandages, once hailed as groundbreaking, were found largely ineffective and abandoned. The same dressing is now commercially available as a topical antimicrobial dressing which has favourable effects on the healing of excision wounds. ChitoFlex® haemostatic dressings are similar to HemCon® but available in stuffable strip form. Chitopack®C is a cotton-type wound dressing commercialised by Eisai Co. Ltd., Japan in 1993; Chitipack®P is a β-chitin-based dressing from the same company; however, it was mainly used for veterinary applications. Chito-Seal® a topical haemostasis pad marketed by Abbott Vascular Inc., and is intended for use in the management of bleeding wounds such as vascular access sites and percutaneous catheters or tubes. The pad is coated with Abbott’s proprietary chitosan gel. When placed over the puncture site, it becomes a powerful cell-binding agent consisting of positively charged chitosan molecules which attract negatively charged red blood cells and platelets, thus accelerating clot formation and haemostasis. Chitopoly (Fujibo Holdings Inc., Japan) is a fabric consisting of chitosan and polynosic, which is antibacterial, an antideodorant and causes no irritation to skin. The TegasorbTM wound dressing by 3M contains chitosan which is used with partial- and full- thickness dermal ulcers, leg ulcers, superficial wounds, abrasions, burns and donor sites. Chitosan interacts with wound fluid and a soft semitransparent absorbent mass is produced which enhances wound healing. The TraumaStatTM haemostatic wound dressing is a unique nonwoven substrate comprised of porous polyethylene fibres which contains a large amount of precipitated silica. This substrate is coated with chitosan which is manufactured from the purest form of chitosan, ChitoClearTM. Clo-Sur® Pad is a nonwoven sealed soluble form of chitosan used as an antimicrobial barrier. This device has received FDA clearance for use in the local management of bleeding wounds such as vascular-access sites. ChiGel is a chitosan-based wound dressing which is soluble in a neutral environment and forms a gel at pH 7.4 and 37 °C. ChiGel has been developed by a company in Israel and indicated for diabetic wound healing, osteoarthritis and rotator cuff. It is employed as a topical therapy and strongly adheres to the wound, which helps stimulate the natural healing process. It will also support the wound’s moist environment while allowing critical gas exchange.
Studies have shown that it stimulates and supports the intrinsic healing process of slow or nonhealing wounds.
8.4.1 Chemical Modifications
Chitin and chitosan are interesting polysaccharides due to the presence of the amino group functionality, which could be suitably modified to impart desired properties and distinctive biological functions, including solubility. Apart from the amino groups, they have two hydroxyl entities which allow effecting appropriate chemical modifications to enhance solubility. The possible reaction sites for chitin and chitosan are illustrated in Figure 8.6 [40]. As with cellulose [41], chitin and chitosan can undergo many chemical modification reactions such as etherification, esterification, crosslinking, graft copolymerisation and so on, which have been summarised by Muzzarelli [42]
and Hon [43]. A number of authors have reviewed the area emphasising various aspects of the potential chemical modification of chitosan. The amino functionality gives rise to chemical reactions such as acetylation, quaternisation, reactions with aldehydes and ketones (to give a Schiff’s base), alkylation, grafting, chelation of metals and so on, to provide a variety of products with properties such as antibacterial, antifungal, antiviral, antiacid, antiulcer, nontoxic, nonallergenic, biocompatible, biodegradable and so on. The hydroxyl functional groups also enable various reactions such as o-acetylation, H-bonding with polar atoms, grafting and so on. Due to the intractability and insolubility of chitin, attention has been given to chitosan with regard to developing derivatives which exhibit well-defined molecular architecture, and have advanced properties and functions.
CH2OH
CH3
NH
HOH2C
H2N CH2OH
H2N HO
HO
O HO
O
O
O
O C=O
O
O
Figure 8.6 Illustration of the possible reaction sites in chitin and chitosan.
Reproduced with permission from C.K.S. Pillai, W. Paul and C.P. Sharma, Progress in Polymer Science, 2009, 34, 7. ©2009, Elsevier [40]
Recently, chitosan and its derivatives have been reported as attractive candidates for scaffolding materials because they degrade as the new tissues are formed, without any inflammatory reactions or toxic degradation [44, 45]. In tissue-engineering applications, the cationic nature of chitosan is primarily responsible for electrostatic
interactions with anionic glycosaminoglycans (GAG), proteoglycans and other negatively charged molecules.
8.4.2 Action of Chitosan
Chitosan is a polymer with a number of basic amino groups and hence possesses an overall cationic charge at acidic pH. This is due to the presence of primary amines on the molecule that bind protons according to Equation 8.1:
Chit − NH2 + H3O+⇔ Chit − NH3+ + H2O (8.1)
The charge density of chitosan has been found to depend on the degree of acetylation (DA) and pH. The amino group in chitosan has a pKa value of ~6.5, which leads to protonation in an acidic solution with the charge density dependent on pH and the %DA value. The amino group becomes deprotonated and chitosan tends to precipitate in a solution of pH greater than 6.5. The multitude of cationic sites along the chitosan chain, formed due to the protonation of amino groups by acids, increases its solubility by increasing both the polarity and the degree of electrostatic repulsion. The wound healing properties of chitin and chitosan are affected by the degree of deacetylation, the molecular weight and physical state. Chitin, chitosan and its derivatives have been reported to accelerate wound healing by enhancing the functions of some inflammatory cells, such as infiltration of polymorphonuclear cells, macrophages, and fibroblasts or osteoblasts.
Malette and co-workers [46] reported that a chitosan solution formed a coagulum when in contact with whole blood and concluded that it is a good substitute for graft haemostasis. The haemostatic property of chitosan involved the agglutination of red blood cells. The polycationic property of chitosan and its nonspecific binding to cell membranes are possibly related to its haemostatic property. Although various applications of chitosan have been reported, at the physiological pH value of 7.4 chitosan is insoluble and ineffective in many cases. The amino groups become deprotonated and the cationic property decreases significantly. The ineffectiveness and failure of some chitosan sponges as a haemostatic bandage could be due to deprotonation. Therefore, various studies have been performed on the development of water-soluble chitosan via chemical modifications and derivatisations. Some of these modifications to chitosan include: sulfonation, N-acetylation, alkylation, hydroxypropyl chitosan, chitosan-saccharide derivatives, O-succinyl-chitosan, quaternisation and carboxymethylation.
8.4.3 Chitosan Derivatives
A simple introduction of chemical groups, such as alkyl or carboxymethyl, into chitin and chitosan can significantly increase their solubility in neutral and alkaline pH values without affecting their chemical and biological characteristics. Carboxymethyl chitosan (CMCh) is a chitosan derivative obtained from the carboxymethylation of chitin with chloroacetic acid in an alkaline solution. Carboxymethyl derivatives of chitin and chitosan are clinically safe [47] and have shown promise in wound healing [48]; they have also been shown to be as efficacious as an alginate dressing in the treatment of partial-thickness skin graft donor sites [49]. A cell migration study demonstrated that the migration of fibroblasts was significantly enhanced by the presence of CMCh in a concentration-dependent manner. CMCh also induced increased proliferation and secretion of three kinds of cytokines and enhanced wound healing compared with the control [50, 51]. NOCC has been shown to be an effective inhibitor of the formation of post-surgical peritoneal adhesion [52], in addition to decreasing adhesion in the healing of colonic anastomosis [53] and reducing intra- abdominal adhesions [54]. A pectin crosslinked CMCh hydrogel has been shown to be a promising wound-dressing material which is nontoxic and blood compatible; this hydrogel can maintain a moist environment which is conducive for wound healing [55]. A composite membrane with chitosan and CMCh was found to be haemostatic, enabled rapid healing and demonstrated histocompatibility, which indicated its usage as a dressing for skin repair; it also had the potential to promote wound healing and inhibit keloid formation [56]. A zinc complex of CMCh has exhibited a significantly high level of antimicrobial activity and can be used as a potential wound-healing device [57]. The CMCh dermal scaffold has been shown to rapidly induce the growth and maturation of blood vessels during wound healing after a burn. It is beneficial for wound repair at an early stage as it inhibits scar proliferation [58]. CMCh hydrogels have been shown to promote cell attachment and the rapid growth of fibroblasts, and have potential as skin scaffolds and wound-healing materials [59].
A photocurable CMCh displayed good wound-healing properties on a burn wound model [60]; it promoted wound healing by activating macrophages, accelerating fibroblast growth and exerting a considerable effect on the secretion of a series of cytokines [61]. Histological studies demonstrated that the NOCC/oxidised alginate hydrogel significantly enhanced the re-epithelialization of the epidermis and collagen deposition in the wound tissue, as well as the process of wound healing [62]. An amphiphatic carboxymethyl-hexanoyl chitosan hydrogel has also been studied as a rapid stem cell delivery system to efficiently enhance corneal wound healing [63].
N-carboxybutyl chitosan membranes have been developed for topical wound- healing applications [64, 65]. N-carboxybutyl chitosan exhibited the formation of regularly organised cutaneous tissue with reduced anomalous healing [66]. Similarly, dibutyryl chitin (DBC) was found to elevate the GAG level in the granulation tissue,
demonstrating its beneficial effect in wound repair [67]. Polyacrylic acid grafted chitin exhibited a faster rate and better pattern of epidermal development, with higher dermal cell proliferation, demonstrating its better efficiency in wound healing compared with Intrasite® [68]; this was similar to the alginate dressing in terms of pain score and wound healing [69]. Developmental chitin fibres have exhibited relatively high keratinocyte and fibroblast cell attachment and spreading compared with commercial chitin fibre (Beschitin®) [70], which is helpful in wound healing and the regeneration of skin. Partially deacetylated chitin hydrochloride exhibited haemostatic properties similar to collagen hydrochloride and has a positive effect in wound healing [71].
Water-soluble chitin (WSC), prepared by controlling the DA and molecular weight, was found to be more efficient than chitin or chitosan as a wound-healing accelerator [72]. WSC treated skin had the highest tensile strength and the arrangement of collagen fibres in the skin was similar to normal skin [73]. DBC is obtained by the reaction of chitin with butyric anhydride in the presence of a catalyst and is easily soluble in organic solvents. Wound dressings made from DBC fibres exhibited an ordered accelerated healing of the wound and the material biodegraded during the healing process [74]. The topical application of DBC significantly reduced patient-assessed skin wound rating scores and increased skin remodelling in an animal model by increasing the expression of type 1 collagen and filaggrin. The study demonstrates that DBC efficiently accelerates the proliferation of HaCaT keratinocytes [75]. In addition, DBC efficiently inhibits inflammation and has potential as an effective anti-inflammatory and wound healing agent [76]; DBC has also been studied as a textile dressing [77]. Satisfactory wound healing in burn wounds and post-operative/
post-traumatic wounds has been achieved clinically [78].
It has been reported that N-sulfosuccinoyl chitosan exhibited significantly higher wound healing activity compared with other chitosan derivatives [79]. Polyelectrolyte complexes of chitosan containing chondroitin-sulfate and hyaluronic acid have been studied for wound-healing applications. However, the performance of these complexes did not exceed that of pure chitosan, although there were no adverse effects [80, 81].
Chitosan modified by ionically binding heparin has shown beneficial effects in wound healing. It is hypothesised that the stimulatory effect of this hydrogel is caused via the stabilisation and activation of growth factors that bind to immobilised heparin, and the increased stabilisation and concentration of endogenous growth factors in the wound area is caused by heparin-chitosan [82, 83]. A water-soluble ointment of a chitosan-heparin complex has previously exhibited increased wound-healing effects [84], with heparin attenuating the effect of chitosan during wound healing [85]. Chitosan, when grafted with polyethylene glycol (PEG) modified tyramine, exhibited enhanced solubility and had the ability to form an in situ curable hydrogel.
This hydrogel showed superior healing effects on skin incisions when compared with sutures, fibrin glue and cyanoacrylate [86]. Cyanoethyl chitosan was prepared by treating activated chitosan with acrylonitrile vapour; the resultant polymer was
soluble in trifluoroacetic acid and was used to make fibres via electrospinning. The mats prepared from these fibres exhibited excellent antibacterial properties and were developed as a possible wound dressing material [87]. Chitosan acetate is the simplest derivative of chitosan and has significant cationic properties. The HemCon® bandage is an engineered haemostatic dressing based on chitosan acetate [88]. Phosphorylated chitin and chitosan have also been reported to have a beneficial application in wound healing [89].