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1. INTRODUCTION The skin is the largest organ in the human body comprising 12-15% of body weight, with a surface area of 1-2 meters. Skin is continuous with, but structurally distinct from mucous membranes that line the mouth, anus, urethra, and vagina. Two distinct layers occur in the skin, namely the dermis and epidermis. The basal cells lie directly on the basal membrane that forms a definite border between the dermis and epidermis. The basal cells acting as mother cells, by cell division, provide for the continuous regeneration of the skin. The basal cell layer is comprised mostly of keratinocytes which are either dividing or non-dividing. In the basal layer are also found the melanocytes, which are the pigment producing cells. The skin functions in thermoregulation, protection, metabolic functions and sensation. The dermis is attached to an underlying hypodermis, also called subcutaneous connective tissue, which stores adipose tissue. The skin is made up of cells like fibroblasts, macrophages and adipocytes embedded in an extra cellular matrix [ECM] consisting of ground substance and fibers formed by collagen and elastin along with glycosaminoglycans [GAG], proteoglycans and glycoproteins. 1.1 WOUNDS Wound can be defined as a disruption of the normal anatomical relationship of tissues as a result of injury. Wounds can be of open and closed type. Open wounds have an opening or break in the skin, whereas in closed wounds, skin is not broken, but the impact from the damaging object would 1 have injured or crushed tissues lying below the tissue point. These wounds are commonly known as bruises and it will be visible due to discoloration resulting from the rupturing of blood capillaries in the injured area. Different types of open wounds are based on the manner in which the skin or tissue is broken and there are six general kinds of wounds: abrasions, incisions, lacerations, punctures, avulsions, and amputations. Many wounds, of course, are combinations of two or more of these basic types (Linares 1996, Calvin 1998). Figure 1.1 Anatomy of skin 1.1.1 Ulcer An ulcer is an open sore of the skin, eyes or mucous membrane, often caused, but not exclusively, by an initial abrasion and generally maintained by an inflammation, infection, and/or medical conditions which impede healing. 2 In other words, it is a macroscopic discontinuity of the normal epithelium. Other causes of skin ulcerations include pressure from various sources and venous insufficiency, which is seen very often in diabetes milletus, resulting in serious pathological conditions. 1.1.2 Burns Burns form the most traumatic of all skin injuries, which may be due to thermal, chemical or electrical means. In burn injuries the trauma and subsequent repair relies greatly on the extension and the depth of the wound. First-degree burns affect the outer layer of the skin, causing pain, redness, and swelling. Second-degree burns affect both the outer and underlying layers of the skin, causing pain, redness, swelling, and blistering. Third-degree burns extend into deeper tissues, causing brown or blackened skin that may be numb (Ho-Asjoe et al 1999). 1.2 WOUND HEALING The process of wound healing involves interaction of a variety of different cell types and matrix components. Various types of cells respond to environmental signals in a specific manner in order to carry out their genetic programme of proliferation, differentiation and function. Cells synthesize different proteins for their proliferation and migration, all of which are controlled in a phased manner. The healing process starts from clotting of blood at the site of wound and gets completed with remodeling of the tissue. However the restoration of the disrupted cellular and anatomic continuity of organisms or its parts by way of healing to ensure wound closure and 3 functional restoration of the damaged tissue is a marvelous process that is not yet fully and clearly understood (Oliver et al 1992). 1.3 PHYSIOLOGICAL BASIS OF WOUND HEALING Wound healing is a fundamental biological process that allows the individual to maintain structural and functional integrity in response to trauma. Healing process is a well-orchestrated cascade of overlapping events with haemostasis, inflammation, proliferation and remodeling phases. After injury the cells release soluble products termed cytokines. Cytokines are polypeptides, which allow communication between cells, causing major changes in cell behavior. These cellular factors initiate events that lead to the subsequent phases of wound healing (Diegelmann and Evans 2004). 1.3.1 Coagulation Platelets agglutinate at the site of injury and a fibrin clot forms by the activation of the coagulation cascade. Thrombin induces platelet degranulation, leading to the release of growth factors viz., platelet derived growth factor (PDGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), epidermal growth factor (EGF) as well as adhesive glycoprotein such as fibronectin. In addition to providing haemostasis, the fibrin clot acts as a matrix for colonization of inflammatory cells, which are attached to the wound site via chemotaxis by PDGF and TGF-α. 4 1.3.2 Inflammation Inflammation, the second phase of wound healing, occurs 1 – 5 days after injury. Migrating inflammatory cells accumulate in the healing wound with neutrophils predominating in the early hours of inflammation. Neutrophils help to decontaminate the wound through phagocytosis of bacteria and foreign bodies, after which macrophages outnumber lymphocytes. Macrophages release chemotactic factors (eg. Fibronectin) to attract fibroblasts to the wound area. New blood vessels growth follows a gradient of angiogenic factors produced by hypoxic macrophages because macrophages do not produce these angiogenic factors when either fully oxygenated or anoxic. Macrophages can be considered as factories for growth factor production, including PDGF, fibroblast growth factor, vascular endothelial growth factor, TGF-β, and TGF-α (Falanga 1993). These cytokines are important in inducing cell migration as well as proliferation and matrix production. Macrophages thus appear to play a pivotal role in the transition between inflammation and repair. 1.3.3 Proliferation The third phase of wound healing is tissue formation, which occurs between 3-12 days. Re-epithelialization begins at the wound edges as early as 24 hours post-injury and granulation of the wound starts around post-injury day 5 for the re-establishment of the integrity of the epidermis and dermis at the wound site. Granulation tissue is formed as macrophages, fibroblasts and endothelial cells move into the wound space. Fibroplasia begins with 5 fibroblast proliferation and collagen deposition stimulated by TGF-β. Wound contraction occurs in full thickness injuries and reaches a peak between 5 and 15 days after injury. Contraction of the wound is mediated by myofibroblasts and this continues through this phase leading to remodeling (Moulin 1995). 1.3.4 Remodeling Tissue starts to reorganize itself as early as day 3 and continues until it returns to a normal tissue structure. During which the accumulation of collagen ceases and net loss of collagen begins by day 21. Although new collagen continues to be deposited, net resorption occurs due to increased degradation of formed collagen by collagenase. As macrophages begin to disappear, angiogenesis and fibroblast proliferation also decrease. (Singer and Clark 1999, Diegelmann and Evans 2004). 1.4 INFECTION – MAJOR CONTRIBUTOR FOR CHRONICITY Among various types of skin wounds, injury due to burns account for more than 60% of morbidity and mortality. Burn wounds represent a breakdown of the skin’s haemostatic function, resulting in fluid and electrolyte loss and infection, which may be life threatening alone or in combination (Tsipouras et al 1995). Although major advances have been achieved in burn wound management and supportive care, infection remains the leading cause of morbidity in burn patients (Greenfield and McManus 1997). A comprehensive review by Bowler (1998), regarding the microorganisms that infiltrate wounds reveal that, aerobic microorganisms, Staphylococcus aureus and Pseudomonas aeruginosa are predominant species 6 responsible for wound sepsis. In addition Escherichia coli, Klebsiella pneumoniae and Streptococcus sp. are other aerobes that contribute to infection in chronic and acute wounds. The progressive bacterial colonization of burns, probably within matter of hours has remained an inseparable phase of healing process. Many times bacterial colonization causes acute wounds to transform into a chronic state, most frequently associated with a predisposing condition that ultimately compromises the dermal and epidermal tissues. During these process bacteria attracts neutrophils through chemo attractants, which in turn engulfs them. The phagocytosed products are secreted out along with a representative tri-peptide called f-met-leu-phe, which in turn gives signals for further recruitment of neutrophils (Tschaikowsky et al 1993). Neutrophils will engorge themselves until they are filled with bacteria and constitute what is called as ‘laudable pus’ in the wound. (Thurston 2000) The heavy infiltration of inflammatory cells further causes prolonged or increased level of endopeptidases, mainly the matrix metalloproteinases (MMPs), resulting in delayed healing. 1.5 ROLE OF MMP IN HEALING PROCESS Matrix metalloproteinases (MMPs) play a major role in healing process, as they are responsible for transformation, organization and maintenance of extracellular matrix. MMPs are zinc-dependent endopeptidases that share common structural and functional elements and are able to cleave one or several extracellular matrix constituents as well as nonmatrix proteins. Ample evidence exists on the role of MMPs in normal and pathological processes including embryogenesis, wound healing, 7 inflammation, and cancer. Endopeptidases modulate the matrix morphology through a highly controlled intracellular and extracellular process. This modulation of ECM is significantly different at various time points during the course of healing for different wound types. Thus it facilitates cellular migration, adhesion, wound contraction and re-epithelialization (Armstrong and Jude 2002). There are various types of MMPs identified which have specific matrix substrates (McCawley and Matrisian 2001) and their activity appears to be controlled at three basic levels, A. Genetic : Controlled through transcriptional factors B. Molecular : Controlled by factors responsible for conversion of proform to active form C. TIMPS : Tissue inhibitors of matrix metalloproteinases (TIMPS) regulate MMP level through feed back inhibition. MMPs are secreted as proenzymes, which become active by removal of propeptide domain. All these MMPs are either bound to cell membrane or secreted into extracellular space. To be secreted they are induced by several factors including PDGF, IL-1, and TNF-α (Mauviel 1993; Kerr et al 1990). Natural inhibitors, TIMPS, growth factor like TGF-β and synthetic inhibitors bring about inhibition of their activity. The activation of the MMPs follows the opening up of catalytic zinc domain. These zinc domains are activated through a “cysteine-zinc” switch mechanism (Figure 1.2). The propeptide 8 forms a cap over the active MMP site with a critical cysteine residue providing the fourth co-ordination ligand to a catalytic zinc ion. Dissociation occurs only at the cysteine–Zn2+ linkage, whereas the Zn2+ complexation with the three histidines is not affected. As a consequence, the MMPs are activated and are now able to degrade appropriate matrix substrate (Kroncke 2001). Figure 1.2 Activation of MMP through Cysteine – Zinc Switch (Kroncke 2001) In general, chronicity of a wound appears to be due to bacterial toxins, which in turn induce elevated MMP expression (Miyajima et al 2001) and imbalance between activation and inhibition (Trengove et al 1999). Thus therapeutic intervention to control infection and to positively regulate MMP balance is considered vital in achieving faster healing of wounds. 9 1.5.1 MMP inhibitors The MMP activity is closely regulated by endogenous inhibitors. The best characterised of these are the tissue inhibitors of metalloproteinases (TIMPs), which comprise a family of four protease inhibitors: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. Their inhibitory activity results from the formation of a non covalent complex of the TIMPs to the C –terminal domain of the proMMPs. Over the past 20 years, impressive efforts have been dedicated to the development of synthetic compounds interacting potently and selectively with the active site of MMPs. Despite these efforts, inhibitors able to differentiate fully one MMP from the others have not been identified so far (Cuniasse et al 2005). The catalytically indispensable zinc ion at the active site has been exploited to design synthetic inhibitors to this enzyme family. In addition, MMPs contain a second zinc atom and atleast two calcium atoms that are not part of the catalytic center but seem to play roles in stabilising tertiary structure (Willenbrock et al 1995). Thus the development of inhibitors for zinc Metalloproteinases has relied on the use of a peptide sequence, recognized by the targeted protease, to which have been grafted different chemical functionalities able to interact potently with the zinc ion of the active-site. This strategy has allowed the identification of several potent pseudo-peptide inhibitors of MMPs. Based on the chemical structure of the zinc-binding group, four different classes of zinc metalloprotease inhibitors have been developed, those incorporating a hydroxamate (CONH–O–), a carboxylate (COO–), a thiolate (S–) or a phosphinyl (PO2–) groups. Hydroxamate exhibited ideal bidentate bonding with the catalytic zinc atom 10 for stable divalent metal coordination within the active site (Whittaker et al 1999). Doxycycline is the only clinically approved drug used to inhibit MMP activity mainly by disrupting the active zinc domains (Nordstrom et al 1998) and is marketed under the name Periostat (Collagenex) for use in the treatment of periodontitis (Peterson 2004). Doxycycline is a member of the tetracycline family of antibiotics and is known to exert biological effects that are independent of its antimicrobial activity. Based on this MMP inhibition activity, doxycycline has been used for many MMP associated disorders like chronic wounds (Chin et al 2003), periodontal disease (Gapski et al 2004), rheumatoid arthritis (Nordstrom et al 1998), corneal erosion (Dursun et al 2001) and skeletal muscle reperfusion injuries (Roach et al 2002). 1.6 WOUND DRESSING Based on the wound type and status, dressing materials should be selected. In general a wound dressing may be of any type, but should possess following basic characteristics (Thomas 1990, Winter 1962). • Maintenance of a moist wound environment • Wound protection from secondary infections by acting as bacterial barrier • Provision of adequate gaseous exchange • Provision of thermal insulation free from particulate or toxin contaminants • Elastic and non-antigenic 11 Availability of wide range of wound dressings till date is probably matched by the diversity in wound types. In addition a wide variety of wound dressings with targeted therapy in mind to address specific problems is emerging in recent days. Food and Drug Administration (FDA 1999) has categorized the type of dressings broadly as – 1. Non-resorbable gauze/sponge dressing for external use, 2. Hydrophilic wound dressing, 3. Occlusive wound dressing, 4. Hydrogel wound and burn dressing and 5. Interactive wound and burn dressings. The choice of dressing is dictated by the type and condition of the wound, is enumerated in Table 1.1 Table 1.1 Types of wound dressings (Young 1997) Wound type Clean, Medium-to-high exudates (epithelialising) Clean, dry, low exudates (epithelialising) Dressing type • • Paraffin gauze Knitted varicose primary dressing • Absorbent perforated plastic filmfaced dressing Vapor-permeable adhesive film dressings • Clean, exudating (granulating) • • • Hydrocolloids Foams Alginates Slough-covered • • Hydrocolloids Hydrogels Dry, necrotic • • Hydrocolloids Hydrogels 12 The design of a wound dressing depends largely on appropriate selection of material. A number of materials have been experimentally and /or clinically studied. Irrespective of the type of material used, it must possess the following characteristics, (Hutmacher 2001) • Three-dimensional and highly porous with an interconnected porous network for cell / tissue growth and flow transport of nutrients and metabolic waste. • Biodegradable or bioresorbable with controlled degradation and resorption rate to match cell / tissue growth in vitro and / or in vivo. • Suitable surface chemistry for cell attachment, proliferation and differentiation • Mechanical properties to match the tissues at the site of implantation and • Easily processed into a variety of shapes and sizes. The application of inert or bioactive synthetic dressing materials remain largely limited to the healing process and minimization of scarring. Similarly moist wound dressings, which possess clinical benefits, pose problems by providing a favorable environment for microbial proliferation (Eaglstein 1993). Among recently available dressings, collagen-based dressings have shown to actively influence the healing process by intervening with various tissue components. Moreover they possess all the characteristics of ideal material for scaffolding as mentioned previously. 13 1.7 BIOMEDICAL POTENTIALS OF COLLAGEN The collagen family has been well described for many years by various authors (Harkness 1966, Prockop and Kivirikko 1995). In humans 25 distinct types of collagen have been identified to date on the basis of protein and / or DNA sequence information (Gelse et al 2003). Collagen is distinct from other proteins as it is composed of three polypeptide chains forming an unique triple helical structure. Organization of individual helices into fibrils and further into fibrillar bundles, results in stable macromolecular structure. Though collagen has phylogenetically well conserved primary sequence and triple helical structure (Tanzer and Kimura 1988), it exhibits structural variations from tissue to tissue with reference to alignment of fibrils, diameter and density of packing. This fibrillar diversity leads to specific mechanical and structural properties, characteristic of each tissue. For example collagen preparation from bovine skin contains 80 - 85% of type I collagen and 1520% of type III collagen and in cartilages and vasculatures type II is present (Kivirikko 1998). As a consequence of this structural and functional significance, collagen can be fabricated into various physical forms (Table 1.2). Thus collagen based wound dressings offer a high degree of plasticity enabling application in wound treatment (Pachence 1996). 14 Table 1.2 Commercially available collagen based dressings (Pachence 1996) Materials Type Partially purified skin Company (Brand name) LifeCell Integra Life Sciences Corp. (Helistat) Collagen sponges Johnson & Johnson (Instat) MedChem (Acti-Foam) BioCore (SkinTemp) Collagen fibers or fleeces Collagen Powder Collagen composite dressing Hydrolyzed Collagen 1.8 Integra LifeSciences Corp. (Helitene) MediChem (Avitene) Medifil (BioCore) Fibracol (Johnson & Johnson) Biobrane (Mylin) Chronicure (Derma Sciences) SHARK SKIN COLLAGEN The molecular structure and biological functions of collagen type I from land animals have been extensively investigated. Bovine and porcine type I collagen provide a readily available source of scaffold materials for various biomedical applications. However these sources have some potential risk of infectious diseases such as bovine spongiform encephalopathy (BSE) or transmissible spongiform encephalopathy (TSE) (Nomura et al 2000). In order to attenuate the risk to manufacture biomaterials an alternate safe collagen must be considered. 15 Type I collagen from aquatic animals may provide an alternative collagen source and shark skin collagen in particular is potentially important. Shark skin, which constitutes about 11% of the body weight, is mainly discarded as industrial waste, except for its use as leather. The main protein in shark skin is type I collagen, the structure basically resembles that from domestic animals and also has the ability of self assembly to form fibrils as that of mammalian type I collagen (Kimura et al 1981). The content of imino acids and the type of cross-linkages vary from mammalian collagen, which reflects the low denaturation temperature of shark skin collagen (Nomura et al 2000). However, the low denaturation temperature can be improved by exogenous crosslinking for biomedical applications. Easy extractability and better yield of collagen are advantageous for applications. Hence, shark collagen is a promising source of type I collagen for various biomedical applications. The milk shark, Rhizoprionodon acutus, is a requiem shark such as the tiger shark, blue shark, bull shark, etc. of the family Carcharhinidae, found in the tropical waters of the Indo-West Pacific and eastern Atlantic oceans between latitudes 35° N and 30° S, from the surface to 200 m. Its length is upto about 1.75 m for a specimen of unknown gender and 80 cm for females. The milk shark is a small shark with a long, narrow, snout, big eyes without notches, long labial furrows, and oblique-cusped teeth, which may be smoothedged or weakly, serrated. The second dorsal fin is small, low and behind the larger anal fin, with no interdorsal ridge. It is found on continental shelves, often on sandy beaches and rarely in estuaries, also reported to enter 16 freshwater and recorded several times from Cambodia as far upstream as the Great Lake. Occurs near the surface in shallow waters, and feeds mainly on small pelagic and benthic bony fishes, also cephalopods and other invertebrates. It is viviparous and the meat is utilized fresh and dried salted for human consumption and for fishmeal. Figure 1.3 Milk shark (Rhizoprionodon acutus) 1.9 CROSSLINKING 1.9.1 Natural crosslinking The systematic packaging of the triple-helices lends strength and resilience to the collagen fibers. Additional mechanical and chemical stability derives from intra- and intermolecular crosslinks. Initially, the formation of crosslinks is mediated by lysyl oxidase during fibril formation (Yamauchi and Mechanic 1988). The enzymatic activity is limited to the non-helical telopeptide regions and leads to the conversion of selective lysyl and hydroxylysyl residues to the corresponding aldehydes allysine and hydroxyallysine. While the fibrils associate, the aldehydes can spontaneously react. Intramolecular crosslinks form between two α-chains in the non-helical 17 section of the same molecule by aldol condensation of two aldehydes (Yamauchi and Mechanic 1988, Reiser et al 1992). Intermolecular crosslinks occur between the telopeptide region of one collagen molecule and the helical region of a quarterly staggered, adjacent molecule. These bridges between two different tropocollagen molecules result from aldimine formation between aldehyde residues and ε-amino groups presented by lysine and hydroxylysine (Reiser et al 1992). The interchain bifunctional crosslinks are still reactive and continue to form polyfunctional crosslinks through multiple condensations with histidine, lysine, or hydroxylysine residues (Graham and Gallop 1994). Hence, through specific self-aggregation and crosslinking, collagen can form fibers of unusual strength and stability, the primary reason for the usefulness of collagen in medical devices. 1.9.2 Exogenous crosslinking Natural crosslinking gives high tensile strength and proteolytic resistance to collagen. Due to dissociation of crosslinks in the course of the above described isolation processes, reconstituted forms of collagen such as films, fibers, or sponges can lack sufficient strength and may disintegrate upon handling or collapse under the pressure from surrounding tissue in vivo. Furthermore, the rate of biodegradation has to be customized based on the specific application. For example as a hemostat, collagen has accomplished its mission once the blood clot has formed, whereas for tissue augmentation an implant has to maintain its scaffolding properties while it is gradually replaced by host collagen (Van et al 1994). Thus, it is often necessary to confer mechanical firmness and collagenase resistance by introduction of 18 exogenous crosslinking into the molecular structure. Several physical and chemical crosslinking methods have been reported. Physical methods include dehydrothermal treatment and UV irradiation (Welz and Ofner 1992). However, these methods cannot control the degree of crosslinking. Chemical methods include formaldehyde, glutaraldehyde, hexamethylenediisocyanate, carbodiimide, polyepoxy compounds and acyl azide. A major handicap of chemical crosslinking agents is the potential toxic effect of residual molecules and / or compounds formed during in vivo degradation (Friess 1998). Glycosaminoglycans (GAGs) are long, unbranched polysaccharides that are a significant component of proteoglycans, do not elicit an immune response on their own, and have been used extensively for tissue engineering applications. Copolymerization of collagen with GAGs has been observed to increase the stiffness and toughness and decrease the degradation rate of collagen (Yannas et al 1975). While the precise mechanism leading to these effects have yet to be elucidated, collagen copolymerization with GAG is used as an alternative to heavy crosslinking of collagen which can often render the material brittle. Scaffolds fabricated from type I collagen and a glycosaminoglycan have been used to study cell migration and contraction in vitro (Ueno et al 2001, Freyman et al 2001, Sethi et al 2002) as well as induce regeneration of the skin, conjunctiva, and peripheral nerves in vivo ( Yannas 1990). Chitosan is a natural polysaccharide, which is structurally similar to GAGs. It consists of β (1-4) linked D-glucosamine residues, and has been reported to be nontoxic, bioabsorbable and to promote wound healing 19 (Francis and Howard 2000). Furthermore, the incorporation of chitosan into a collagen scaffold is known to increase its mechanical strength, as it forms an ionic complex between the positively charged chitosan and the negatively charged collagen (Taravel and Domard 1996). Chitosan has also been reported to stimulate the activity of growth factors (Ueno et al 2001) and to contribute to the maintenance of the chondrogenic phenotype, especially in terms of its morphology (Sechriest et al 2000). One of the interesting features of chitosan is its cationic nature occurring from primary amine groups, thus providing a high charge density in an acidic solution. The cationic charges allow chitosan to form water-insoluble ionic complexes with a variety of polyanionic substances such as sulfate, citrate, and tripolyphosphate (TPP) for drug delivery applications (Shu and Zhu 2002). Furthermore, this characteristic can be used for the delivery of biologically active polyanions such as DNA (Lee et al 1998, Roy et al 1999). 1.10 COLLAGEN BASED DRUG DELIVERY SYSTEMS Major advantages using collagen as a biomaterial for development of delivery systems are – available in abundance and easily purified, nonantigenic, biodegradable and bioresorbable, non-toxic and biocompatible, synergic with bioactive components, high tensile strength, easily modifiable as desired by utilizing functional groups comparable with synthetic polymers (Werkmeister and Ramshaw 1992). The use of collagen as a drug delivery system is very comprehensive and diverse (Friess 1998). Table 1.3 enumerates the types of collagen drug delivery applications. Any preparation that uses collagen as a matrix may act as (a) Reservoir – increase contact time 20 with the host, (b) Reversible – bind drug such that they are released in a delayed mode, (c) Reduce the likelihood of systemic toxicity. Most of the delivery devices mentioned have been designed to sustain the release of therapeutics. Controlled delivery is essential for drugs, which have narrow line of demarcation between therapeutic and toxic concentration. An extensive compilation on different types of collagen based delivery systems for varied applications, including antibacterial, recombinant proteins, growth factors and gene therapy has been comprehensively described (Ruszczak and Friess 2003). 1.11 LOCAL DELIVERY THERAPEUTICS FOR WOUND HEALING The importance of drug delivery is not only pertinent to physiological response but also to the mechanism of drug release, especially as a carrier for on-site delivery of antibacterials. The scaffolds used for this purpose are thin films, gels, sponges and bilayered dressings with or without chemical modifications (Ruszczak and Friess 2003). Fibrillar collagen matrices alone are capable of only moderating the release of drugs hence a significant nonfibrillar collagen is necessary to modulate the diffusive parameters. Further chemical modifications like succinylation, electrostatic charge interactions are some of the strategies adopted to improve the release pattern of drugs. In this category several wound healing materials like composite collagen – chondrotin 4 and 6 sulphate (Berthod et al 1994), polyurethane coated atelocollagen – rifampicin composite (Suh et al 1994) materials, bilayered dressing with carboxymethyl – chitin hydrogel materials and chitosan acetate 21 thin films were able to sustain the antibiotic delivery. Emerging systems deliver growth factors either through implants or through bioactive thin films (Royce et al 1995, Mao et al 2005). Though collagen alone can be tailored into various forms to deliver therapeutics, many of these systems are unable to control the release pattern in a steady state for prolonged time (Friess 1998). Table 1.3 Applications of collagen based drug delivery system (Ruszczak 2003) Application mode Drug Indications erythromycin, Infection, glaucoma Pilocarpine, gentamycin, Gentamycin, vancomycin, tobramycin, Polymyxin B, Infection, glaucoma trimethoprim, amphotericin B, 5Shields FU, pilocarpine, steroids, Glaucoma, Inflammation flurbiprofene, Particles Cyclosporine Allograft implication Gels Keterolac Inflammation 111 Vinblastine, cisplatine, 5-FU, In, 90 Y labeled monoclonal antibodies, Cancer Treatment Aqueous injection TGF-β, FGF, Insulin, Growth Wound Repair Hormone Gentamycin, cefotaxim, fusidic acid, clindamycin, all-trans Infection, cervical dysplasia Sponges retinoic acid, Growth factors, bone Tissue regeneration morphogenic proteins Medroxyprogestrone acetate, human growth hormone, Tissue repair and Films immunostimulants, tetracycline, regeneration growth factors Retinol, tretinoin, tetracain, Local anaesthetic, dermal Microparticles lidocaine, ethracridine lactate Minocycline, lysozyme, Infection and Wound Monolithic devices intereukin-2, interferon Repair Inserts 22 To accomplish controlled release, drugs can be entrapped in micro-carriers like micro/nano spheres, liposomes or as microencapsules. The following section explains in detail the advantages of microspheres in controlling the release pattern and the polymers utilized to develop microcarriers. 1.12 IMPACT OF CONTROLLED DELIVERY DEVICES IN WOUND HEALING The term ‘Controlled Release’ has become associated with those systems from which therapeutic agents may be delivered at a predetermined rate over long time. These include situations requiring the slow release of water-soluble drugs, drug delivery to specific sites and release of two or more agents with same formulations. Among the recent advancements in wound care management, slow release of drugs exhibited by hydrogels provide scope for developing novel wound dressings (Kim et al 1992, Peppas et al 2000, Lay-Flurrie 2004). The ideal drug delivery system should be inert, biocompatible, mechanically strong, and comfortable for the patients, capable of achieving optimum drug loading, simple to administer and remove, safe from accidental release, and easy to formulate and sterilize. Figure 1.4 represents a comparison between release profiles obtained with traditional dosage form and controlled release dosage system. In traditional drug administration the blood level of the agent should remain between a maximum value, which may represent a toxic level, and a minimum value, below which the drug is no longer effective. In controlled drug delivery systems designed for long-term administration, the drug level in the blood remains constant, between the desired maximum and minimum, for an extended period of time (Allen et al 2005). 23 Figure 1.4 Comparison between traditional dosage form and controlled drug release system (Allen et al 2005) 1.13 CONTROLLED RELEASE MECHANISM There are three primary mechanisms by which active agents can be released from a delivery system: diffusion, degradation, and swelling followed by diffusion. Any or all of these mechanisms may occur in a given release system. Diffusion occurs when a drug or other active agent passes through the polymer that forms the controlled-release device. Figure 1.5.1 reference shows the general mode of drug release through diffusion, either on 24 a macroscopic scale, as through pores in the polymer matrix on a molecular level, by passing between polymer chains. Figure 1.5.2 shows the type of system (reservoir system) most widely used for oral delivery of drugs. Figure 1.5.3 shows a typical, dermal and/or the trans-dermal system used in delivering drug to the wound site. In this type of system a reservoir of solid drug, or dilute solution, or highly concentrated drug solution within a polymer matrix is surrounded by a film or membrane of a rate controlling material by which a fairly sustained rate of drug release can be maintained. In order to achieve controlled release of drug in a desired rate, the most widely used systems are swelling-controlled release devices. These systems are initially dry and, when placed in the body will absorb water or other body fluids and swell. The swelling increases the aqueous solvent content within the formulation as well as the polymer mesh size, enabling the drug to diffuse through the swollen network into the external environment. This may be achieved through two phenomena – by diffusion through the polymer matrix (Figure 1.5.4) and matrix swelling-controlled release systems (Figure 1.5.5). Other systems used for controlled delivery of drugs include biodegradable systems in which polymer degrades in the body, environmentally responsive systems, and externally triggered controlled delivery systems (Robinson and Lee 1987, Peppas et al 1997). 1.14 BIOMATERIALS FOR DEVELOPING CONTROLLED RELEASE SYSTEMS Wide ranges of materials (both synthetic and natural) are available for developing controlled release systems. Natural polymers have gained special 25 interest over synthetic polymers mainly because of better biocompatibility, degradation products being biocompatible (in contrast to some of the synthetic monomer), cost effectiveness, and bulk availability. The role of natural polymers in biomedical application is pivotal, especially hydrogels, carrier systems, wound dressings, oral dosage forms, and implants. Biopolymers from marine sources have been studied and utilized in pharmaceutical and biotechnological product development for a number of years (Peppas et al 1997), particularly chitosan as biodegradable biopolymers in drug delivery systems (Skaugrud et al 1999). Figure 1.5 Schematic representation of controlled drug release mechanism through polymer matrix (Peppas et al 1997) 26 1.15 CHITOSAN – BIOMEDICAL AND PHARMACEUTICAL APPLICATIONS This class of marine polysaccharides possesses wide range of biomedical and pharmaceutical applications owing to their excellent biocompatibility and biodegradability (Muzzarelli et al 1988). Chitosan is a linear, randomly distributed, binary heteropolysaccharide consisting of (1–4)linked 2-acetamido-2-deoxy-β-D-glucose and 2-amino-2-deoxy-β-D-glucan units (Figure 1.6). It is derived by alkaline N-deacetylation of chitin, obtained from marine crustaceans. Based on degree of N-deacetylation and molecular weight, it’s physicochemical (e.g. solubility, viscosity), biological (e.g. bioadhesion, absorption enhancement) and formulation properties can be tailored accordingly. Networks formed by ionic crosslinking (Figure 1.7) of chitosan are mainly used for drug delivery. They generally exhibit pH-sensitive swelling and drug release by diffusion through their porous structure (Ruel-Gariepy et al 2000, Remunan-Lopez and Bodmeier 1997). Hence the micro and nano carriers of chitosan are widely used for controlled delivery of wide range of therapeutics. The carriers can be developed by various techniques like emulsion cross-linking, coacervation/precipitation, spray drying, ionic gelation and sieving method. Among these, ionic gelation is preferred for delivering therapeutics required to be delivered for a short time period in a controlled fashion. Especially, in cases where drug concentration has to be achieved in a short duration (burst release) and further to be maintained in a steady state. This strategy is most suitable for materials intended for wound healing application. Apart from that, it is a Glycosaminoglycan (GAG) having natural ability to interact with host cells, 27 and also similar to the integral component of ECM (Agnihotri et al 2004, Berger et al 2004). The advancement in wound dressing strategies, specificities and availability of wide variety of biomaterials provide impetus in developing wound dressings for our desired need. Implication of controlled drug delivery phenomenon along with the understanding of wound healing phases make it possible to design scaffolds to tackle wounds of varied degree of pathology. Figure 1.6 Structural unit of chitosan (Agnihotri et al 2004) Figure 1.7 Schematic representation of ionically crosslinked chitosan (Berger et al 2004). 28 AIM OF THE WORK To develop collagen based controlled delivery system for wound healing, with the main emphasis on mitigating infection and to hasten healing with balanced remodeling. Collagen, especially in the form of membranes/ films has gained more importance in recent past, particularly for its healing properties. Biomedical use of bovine and porcine skin type I collagen have been well documented. But due to potential risk of Bovine spongiform encephalopathy (BSE) and Transmissible spongiform encephalopathy (TSE) there is an increasing demand for an alternate source especially collagen from aquatic animals like squids and fish. Squids are more glycosylated and the collagen yield is low. The collagen yield from fish skin is high and rich in type I collagen but the disadvantage is the presence of scales. Whereas shark could be potential alternative because shark skin is treated as waste and has very less and tiny scales and the collagen extractability is high. Collagen and Glycosaminoglycan (GAG), identical constituents of native tissues, are widely utilized to fabricate scaffolds serving as an active analog of extracellular matrix (ECM). The combination of collagen and GAG is unique in skin regeneration since collagen or GAG alone cannot heal full thickness wounds. Chitosan and Aloe vera which are GAG analogs have numerous biomedical applications. Therefore, a reconstituted shark skin type I collagen composite scaffold with chitosan or Aloe vera impregnated with doxycycline loaded chitosan microspheres was developed, characterized and evaluated in vivo. 29 The present work encompasses the following goals: • Isolation and characterization of collagen from the skin of milk shark (Rhizoprionodon acutus). • Development of reconstituted shark skin collagen scaffolds incorporated with chitosan and parenchymal Aloe extract and their characterization, including assessment of physicochemical, morphological and biocompatible properties. • Development of doxycycline hyclate loaded chitosan microspheres, and drug loaded microsphere incorporated collagen scaffold, and assessment of morphological, antibacterial and in vitro release studies. • Evaluation of the efficiency of doxycycline hyclate loaded chitosan microspheres impregnated collagen scaffold against infection challenged burn wound in rat model. Assessment of its efficiency through healing rate, decrease in microbial load, collagen and Glycosaminoglycan (GAG) turn over, expression of proinflammatory cytokines (IL-6, IL-1β and TNF-α) and Matrix Metalloproteinases (MMPs) expression, and efficient remodeling. 30