Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications Jonathan J. Elsner, Israela Berdicevsky, Adaya Shefy-Peleg and Meital Zilberman Abstract There are various wounds with tissue loss. These include burn wounds, wounds caused as a result of trauma, diabetic ulcers and pressure sores. Every year in the United States more than 1.25 million people experience burns and 6.5 million experience various chronic skin ulcers. In burns, infection is the major complication after the initial period of shock and it is estimated that about 75% of the mortality following burn injuries is related to infections. Wound dressings aim to restore the milieu required for skin regeneration by protecting the wound from environmental threats, including penetration of bacteria, and by maintaining a moist healing environment. A wide variety of wound dressing products targeting various types of wounds and different aspects of the wound healing process are currently available on the market. Ideally, a dressing should be easy to apply and remove, and its design should meet both physical and mechanical requirements; namely water absorbance and transmission rate, handleability and strength. Although silver-eluting wound dressings are available for addressing the problem of infection, there is growing evidence of the deleterious effects of such dressings in delaying the healing process due to cellular toxicity. In this chapter wound dressings with controlled release of bioactive agents are discussed. Our novel biodegradable antibiotic-eluting wound dressings are described in details and the engineering aspects in the design are emphasized. The composite material which is based on a biodegradable fibrous polyglyconate mesh bonded with a porous Poly(DL-lactic-co-glycolic acid) matrix, is designed to protect the wound until it is no longer needed, after which it dissolves away by chemical degradation into J. J. Elsner
A. Shefy-Peleg
M. Zilberman (&) Department of Biomedical Engineering, Tel-Aviv University, 69978 Tel-Aviv, Israel e-mail: meitalz@eng.tau.ac.il I. Berdicevsky Department of Microbiology, Technion – Israel Institute of Technology, 32000 Haifa, Israel Stud Mechanobiol Tissue Eng Biomater (2011) 8: 3–37 DOI: 10.1007/8415_2011_66 Ó Springer-Verlag Berlin Heidelberg 2011 Published Online: 16 February 2011 3 4 J. J. Elsner et al. non-toxic products. These new composite wound dressings are advantageous in that they provide better protection against infection, enable faster wound healing and reduce the need for frequent dressing changing. 1 Introduction The skin is regarded as the largest organ of the body and has many different functions. Wounds with tissue loss include burn wounds, wounds caused as a result of trauma, diabetic ulcers and pressure sores. The regeneration of damaged skin includes complex tissue interactions between cells, extracellular matrix (ECM) molecules and soluble mediators in a manner that results in skin reconstruction. The moist, warm, and nutritious environment provided by wounds, together with diminished immune functioning secondary to inadequate wound perfusion, may allow build-up of physical factors such as devitalized, ischemic, hypoxic, or necrotic tissue and foreign material, all of which provide an ideal environment for bacterial growth [34]. Infection is defined as a homeostatic imbalance between the host tissue and the presence of microorganisms at concentrations that exceeds 105 organisms per gram of tissue or the presence of beta-hemolytic streptococci [74, 82]. The main goal of treating the various types of wound infections should be to reduce the bacterial load in the wound to a level at which wound healing processes can take place. Otherwise, the formation of an infection can seriously limit the wound healing process, can interfere with wound closure and may even lead to bacteremia, sepsis and multisystem failure. Evidence of bacterial resistance is on the rise, and complications associated with infections are therefore expected to increase in the general population. Various wound dressings aim to restore the milieu required for skin regeneration and to protect the wound from environmental threats and penetration of bacteria. Although traditional gauze dressings offer some protection against bacteria, this protection is lost when the outer surface of the dressing becomes moistened by wound exudates or external fluids. Furthermore, traditional gauze dressings exhibit low restriction of moisture evaporation which may lead to dehydration of the wound bed. This may lead to adherence of the dressing, particularly as wound fluid production diminishes, causing pain and discomfort to the patient during removal. Most modern dressings are designed according to the well-accepted bilayer structural concept: an upper dense ‘skin’ layer to prevent bacterial penetration and a lower spongy layer designed to adsorb wound exudates and accommodate newly formed tissue. Unfortunately, dressing material adsorbed with wound discharges provides conditions that are also favorable for bacterial growth. This has given rise to a new generation of wound dressings with improved curative properties that provide a local antimicrobial effect by eluting various germicidal compounds. Local delivery of antibiotics and disinfectants addresses the major disadvantages of the systemic approach, namely poor penetration into ischemic and Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 5 necrotic tissue typical of post-traumatic and postoperative tissue, renal and liver complications, and need for hospitalized monitoring [59, 65] by maintaining a high local antibiotic concentration for an extended duration of release without causing systemic toxicity [26, 70, 84]. The effectiveness of such devices is strongly dependent on the rate and manner in which the drug is released [81]. These are determined by the host matrix into which the antibiotic is loaded, the type of drug/ disinfectant and its clearance rate. If the agent is released quickly, the entire drug could be released before the infection is arrested. If release is delayed, infection may set in further, thus making it difficult to manage the wound. The release of antibiotics at levels below the minimum inhibitory concentration (MIC) may lead to bacterial resistance at the release site and intensify infectious complications [24, 25]. A local antibiotic release profile should therefore generally exhibit a considerable initial release rate in order to respond to the elevated risk of infection from bacteria introduced during the initial shock, followed by a sustained release of antibiotics at an effective level, long enough to inhibit latent infection [65]. This chapter introduces types of cutaneous wounds and then describes the main features of wound dressings based on both synthetic and natural polymers. Part of it then focuses on our new concept of antibiotic-eluting composite structures for wound healing applications. 2 Cutaneous Wounds The primary function of the skin is to serve as a protective barrier against the environment. The skin has two anatomic layers, each with a separate function. The superficial, epidermal layer is a barrier to bacteria and vapor (moisture loss). The dermal layer deep to the epidermis provides protection from mechanical trauma and the elasticity and mechanical integrity of the skin. Blood vessels providing nutrition to the epidermal layer run within the dermis. After skin loss, epidermal cells regenerate from deep within dermal appendages, such as hair follicles and sweat glands. The skin’s function as a barrier to infection and fluid loss is lost with injury. Loss of the integrity of large portions of the skin as a result of injury or illness may lead to major disability or even death. Every year in the United States more than 1.25 million people experience burns and 6.5 million experience chronic skin ulcers caused by pressure, venous stasis, or diabetes mellitus [69]. The treating physician must compensate with appropriate fluid management and local wound care. 2.1 Burns It is estimated that 1.25 million burn injuries occur every year in the US. Approximately 450,000 of these cases visit hospital emergency departments and 6 J. J. Elsner et al. 4,500 die annually of thermal related injuries (American Burn Association). Burn injuries are among the most complex and harmful physical injuries to evaluate and manage. The multiple treatment algorithms for burn wounds and resuscitation require quantification of the extent of the burn. Although many new modalities are becoming available to assess burn depth, such as laser Doppler and dielectric measurements, assessment by an experienced practitioner is currently the most reliable judge of burn depth. A simplistic description of burn type and depth is presented in Table 1. The two most important problems encountered clinically with burn victims are dehydration and infection. Despite considerable advancements in burn wound care and infection control practices, infection remains the leading cause of death in this group of patients an it is estimated that about 75% of the mortality following burn injuries is related to infections rather than to osmotic shock and hypovolemia [38, 61]. Clearly the best coverage for wounds requiring serious reconstruction is natural skin taken from the individual himself (an autograft). However, in clinical practice this is not always possible, particularly in large total body surface area burns, as there is often an insufficient amount of skin for autografting available at the time of burn excision, or the physiological condition of the patient precludes the harvesting of skin. Allografts and xenografts can be used to provide temporary wound coverage, but there are issues with graft rejection, availability, and the possibility of disease transfer. Although burn wound surfaces are sterile immediately following thermal injury, colonization with autogenous microorganisms (originating from the skin, gastrointestinal and respiratory flora) or through contact with the contaminated environment (water, air, and healthcare workers) generally occurs within 48 h [1, 20]. The typical burn wound is initially colonized predominantly with Gram-positive organisms, which are replaced by antibiotic-susceptible Gram-negative organisms within approximately 1 week after the burn injury. If wound closure is delayed and the patient becomes infected, thus requiring treatment with broad-spectrum antibiotics, these floras may be replaced by yeasts, fungi, and antibiotic-resistant bacteria [68]. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are the most frequently isolated organisms in most burn units [68]. Systemic treatment against infection is limited by inadequate wound perfusion which restricts migration of host immune cells and the delivery of antimicrobial agents to the wound. In this case the local concentration of the antibiotics may be insufficient and may lead to bacterial resistance. The widespread application of a topical antimicrobial agent on the open burn wound surface can substantially reduce the microbial load and risk of infection [50]. However, it requires frequent changes of the dressing material which causes inconvenience to the patient and comprises a financial burden to the healthcare system. Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 7 Table 1 Classification of burns based on depth characteristics [27, 77] Burn severity Cause/appearance First Caused by flame flash or degree ultraviolet exposure. Red, swollen, and painful. The burned area whitens (blanches) when lightly touched but does not develop blisters Epithelium is intact Second Caused by scald (spill), flame, degree oil, grease. Pink or red, swollen, and painful, blisters that may ooze a clear fluid. The burned area may blanch when touched. Loss of the epidermis and a portion of the dermis. Third Scald (immersion), flame, degree steam, oil, grease, chemical, high-voltage electricity Usually not painful. skin may be red, white, waxy or charred black Loss of the tissue through the dermis including the hair follicles and sweat glands and extending into the hypodermis Notable absence of tissue edema compared with surrounding seconddegree burned area Fourth Direct exposure of skin to degree open flame. Electrical, electrical shock Treatment Healing time Require no specific care Necrotic epidermis will generally slough within 1 or 2 days, revealing intact epidermis Superficial burns generally Superficial burns heal within 10–14 days. heal spontaneously with Deep dermal burns local wound care. usually take more than Deep burns should be rd 3 weeks to heal. treated like 3 degree burns Surgical debridement (removal of dead skin). Treatment with skin graft or other skin replacement. Small third-degree wounds (those of\2 cm) may heal spontaneously by ingrowth from the wound edges, requiring 6 weeks, and the wound may never heal completely and always result in a generous scar. Larger wounds will not heal without surgery Full thickness wound extending through the subcutaneous soft tissue to tendon, muscle or bone Associated with limb loss or the need for complex reconstruction 8 J. J. Elsner et al. 2.2 Ulcers Associated with Pressure and Arterial and Venous Diseases An ulcer is an area of loss of the epithelium, with acute or chronic inflammation in the underlying connective tissue. In an acute ulcer, the epithelium is lost and there is edema, congestion, and polymorphonuclear leukocyte infiltration in the underlying tissue. In a chronic ulcer, there may be exuberant proliferation of young capillaries with plump fibroblasts and chronic inflammatory cells including lymphocytes and macrophages (granulation tissue). Ulceration of the lower limb affects 1% of the adult population and 3.6% of people older than 65 years [46]. Venous disease, arterial disease, and neuropathy cause over 90% of lower limb ulcers. Venous ulcers most commonly occur above the medial or lateral malleoli. Arterial ulcers often affect the toes or shin or occur over pressure points. Neuropathic ulcers tend to occur on the sole of the foot or over pressure points. Apart from necrobiosis lipoidica, diabetes is not a primary cause of ulceration but often leads to ulceration through neuropathy or ischaemia, or both. Ulcers are usually treated with elastic compression bandaging with a simple non-adherent dressing underneath. The bandages should be changed once or twice a week, and the healing rate depends on the initial size of the ulcer, but 65–70% of ulcers heal within 6 month [46]. 2.3 The Biology of Wound Healing Wound healing is a dynamic, interactive process involving soluble mediators, blood cells, ECM, and parenchymal cells. Wound healing has three phases: inflammation, tissue formation, and tissue remodeling—that overlap in time [4, 28]: (i) Inflammation The first stage of wound repair occurs immediately after tissue damage, and components of the coagulation cascade, inflammatory pathways and immune system are needed to prevent ongoing blood and fluid losses, to remove dead and devitalized (dying) tissues and to prevent infection. Haemostasis is achieved initially by the formation of a platelet plug, followed by a fibrin matrix, which becomes the scaffold for infiltrating cells as demonstrated in Fig. 1a. Neutrophils are then recruited to the wound in response to the activation of complement, the degranulation of platelets and the products of bacterial degradation. (ii) New tissue formation The second stage of wound repair occurs 2–10 days after injury and is characterized by cellular proliferation and migration of different cell types. The first event is the migration of epithelial cells and fibroblasts to the injured area to replace damaged and lost tissue. These cells regenerate from the margins, rapidly growing over the wound under the dried scab (clot) (Fig. 1b). Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 9 Fig. 1 Schematic representation of the phases of wound healing: a infiltration of neutrophils into the wound area, b invasion of wound area by epithelial cells, c epithelium completely covers the wound, and many of the capillaries and fibroblasts, formed at early stages have all disappeared [28] The proliferative phase occurs almost simultaneously or just after the migration phase (Day 3 onwards) and basal cell proliferation, which lasts for between 2 and 3 days. Granulation tissue is formed by the in-growth of capillaries and lymphatic vessels into the wound and collagen is synthesized by fibroblasts giving the skin strength and form. By the fifth day, maximum formation of blood vessels and granulation tissue has occurred. Further epithelial thickening takes place until collagen bridges the wound. The fibroblast proliferation and collagen synthesis 10 J. J. Elsner et al. continues for up to 2 weeks by which time blood vessels decrease and oedema recedes. In the later part of this stage, some of the fibroblasts are stimulated by macrophages differentiate into myofibroblasts. Myofibroblasts are contractile cells that, over time, bring the edges of a wound together. Fibroblasts and myofibroblasts interact and produce ECM, mainly in the form of collagen, which ultimately forms the bulk of the mature scar. (iii) Remodeling Remodeling begins 2–3 weeks after injury and lasts for a year or more. During this stage, all of the processes activated after injury wind down and cease. Most of the endothelial cells, macrophages and myofibroblasts undergo apoptosis or exit from the wound, leaving a mass that contains few cells and consists mostly of collagen and other extracellular-matrix proteins (Fig. 1c). In addition, over 6–12 months, the acellular matrix is actively remodeled from a mainly type III collagen backbone to one predominantly composed of type I collagen. This process is carried out by matrix metalloproteinases that are secreted by fibroblasts, macrophages and endothelial cells, and it strengthens the repaired tissue. However, the tissue never regains the properties of uninjured skin. 3 Wound Dressings with Controlled Release of Antibacterial Agents A range of dressing formats based on films, hydrophilic gels and foams are available or have been investigated [86, 88]. Films and gels have a limited absorbance capacity and are recommended for light to moderately exudating wounds, whereas foams are highly absorbent and have a high water vapor transmission rate and are therefore considered more suitable for wounds with moderate to heavy exudation [4]. The characteristics of the latter are controlled by the foam texture, pore size, and dressing thickness. 3.1 Wound Dressings Based on Synthetic Polymers Wound dressings that provide an inherent antimicrobial effect by eluting germicidal compounds have been developed to respond to the aforementioned problems associated with conventional topical treatments with ointments and creams. Wound dressings that incorporate iodine (IodosorbÒ by Smith & Nephew), chlorohexidime (BiopatchÒ by Johnson & Johnson) or most frequently silver ions (e.g., ActicoatÒ by Smith & Nephew, ActisorbÒ by Johnson & Johnson and AquacelÒ by ConvaTec) as active agents are available on the market. Such dressings are designed to provide controlled release of the active agent through a slow but sustained release mechanism which helps avoid toxicity yet ensures delivery of a therapeutic dose to the wound. ActicoatÒ (Smith and Nephew), for instance, is a Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 11 3-ply gauze dressing made of an absorbent rayon polyester core, with upper and lower layers of a nano-crystalline silver-coated high density polyethylene mesh. It is applied wet and is then moistened with water several times daily to allow the release of the silver ions so as to provide an antimicrobial effect for 3 days [21]. Despite frequent usage, there is growing evidence that silver is highly toxic to keratinocytes and fibroblasts and may delay burn wound healing if applied indiscriminately to healing tissue areas [7, 13, 57]. In order to address this issue, the silver in ActisorbÒ (Johnson & Johnson) is impregnated into an activated charcoal cloth, after which it is encased in a nylon sleeve which does not enable the silver in the product to be freely released at the wound surface but nevertheless eradicates bacteria that adsorb onto the activated charcoal component. Another substantial disadvantage of the majority of the available synthetic wound dressings is the fact that similarly to textile wound dressings, the necessary change of dressings may be painful and increases the risk of secondary contamination. Bioresorbable dressings may successfully address this shortcoming, since they do not need to be removed from the wound surface once they have fulfilled their role. Biodegradable film dressings made of synthetic lactide-e-caprolactone copolymers [35] are available for clinical use under the brand names of TopkinÒ (Biomet, Europe) and OprafolÒ (Lohmann & Rauscher, Germany). During the hydrolytic degradation process the pH shifts towards the acidic range, with pH values as low as 3.6 measured in vitro [35]. Although these two dressings do not contain antibiotic agents, it is claimed that the low pH values induced by the polymer’s degradation help reduce bacterial growth [78] and also promote epithelialization [14]. Furthermore, local lactate concentrations may stimulate local collagen synthesis [32]. Film dressings are better suited for small wounds, since they lack an absorbing capacity and are impermeable to water vapors and gases, which may cause accumulation of wound fluids on larger wound surfaces. 3.2 Wound Dressings Based on Natural Polymers Only a handful of natural materials: collagen [66], chitosan/chitin [48] and alginate [75] are already available on the market as either main or additional components to the dressing structure which are able to impact the local wound environment beyond moisture management and to elicit a cellular response. Collagen is the main structural protein of the ECM, and was one of the first natural materials to be utilized for skin reconstruction and dressing applications. Collagen-based products have been available commercially for over a decade. They come in a variety of setups ranging from gels, pastes and powders to more elaborate sheets, sponges, and composite structures [66]. Biological materials such as collagen and chitosan have been reported to perform better than conventional and synthetic dressings in accelerating granulation tissue formation and epithelialization [8, 64, 71]. Chitosan has also been documented as displaying considerable intrinsic antibacterial activity against a broad spectrum of bacteria [51]. 12 J. J. Elsner et al. Gentamicin-eluting collagen sponges have been found useful in both partial-thickness and full-thickness burn wounds. CollatampÒ (Innocoll GmbH, Germany), SulmycinÒ-Implant (Schering-Plough, USA) and SeptocollÒ (Biomet Merck, Germany) are examples of such products which have been found to accelerate both granulation tissue formation and epithelialization. Because these products elute gentamicin, they also protect the recovering tissue from potential infection or re-infection [64, 65]. A comprehensive clinical study of gentamicincollagen sponges demonstrated their ability to induce high local concentrations of gentamicin (up to 9,000 lg/mL) at the wound site for at least 72 h while serum levels remained well below the established toxicity threshold of 10–12 lg/mL [65]. However, the release of antibiotics directly from natural polymers suffers from several disadvantages. First, most natural polymers are hydrophilic and cannot counteract rapid release of the small antibiotic molecules upon water uptake, unless they are highly cross-linked. Second, natural polymers undergo in vivo degradation by proteases. The incorporated drug is thus released by a combination of diffusion and natural enzymatic breakdown of the protein, and is dependent on the biochemical wound setting. Consequently, the active agent is rapidly released from these materials [39, 71]. Simple collagen sponge entrapment systems are characterized by high drug release upon the wetting of the sponge, typically within 1–2 h of application. Sripriya et al. [71] have suggested improving the release profile of such systems by using succinylated collagen which can create ionic bonds with the cationic antibiotic ciprofloxacin so as to restrain its diffusion. It is claimed that in this way ciprofloxacin release corresponds to the nature of the wound in line with the amount of wound exudates absorbed in the sponge. Effective in vitro release from their system was found to last 5 days, and was proven successful in controlling infection in rats. Other studies have aimed to better control drug release or improve wound healing properties by combining collagen with other synthetic or natural biodegradable elements. Prabu et al. [58] focused on achieving a more sustained release of the antimicrobial agent and described a dressing made from a mixture of collagen and PCL loaded with gentamicin and amikacin, whereas Shanmugasundaram et al. [67] chose to impregnate collagen with alginate microspheres loaded with the antibacterial agent silver sulfadiazine (AgSD). Other studies which focused on improving wound healing capabilities tried to incorporate tobramycin, ciprofloxacin [56] and AgSD [44] into collagen– hyaluronan based dressings. The two latter studies did not show conclusive evidence of improved healing properties compared to their control. However, hyaluronan, a structure-stabilizing component of the ECM, is thought to play a role in several aspects of the healing process with hyaluronan-based dressings, and exhibited promising results in the management of chronic wounds such as venous leg ulcers [11, 76]. A wide range of studies describe the employment of the polysaccharide chitosan and its partially deacetylated derivative chitin as structural materials analogous to collagen for wound dressings. Both materials offer good wound protection and have also been found to promote wound healing without excessive Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 13 granulation tissue and scar formation [10]. Chitosan has also been documented as displaying considerable intrinsic antibacterial activity against a broad spectrum of bacteria [51]. Ignatova et al. [33] reported the electrospinning of chitosan with PVA into non-woven nano-fiber mats with good in vitro bactericidal activity against S. aureus and Escherichia coli (E. coli). Another interesting fibrous form which combines the polysaccharides chitosan and alginate was reported by Knill et al. [41], who developed a composite structure of calcium alginate filaments coated with chitosan, utilizing the cationic interaction of chitosan with the anionic nature of alginate to bond the two together. It has been suggested that the core alginate fiber may manage excess exudates whereas chitosan would provide antibacterial, haemostatic and wound healing properties. In this case too, antibacterial testing of the fibers demonstrated an antibacterial effect. Several attempts to improve the chitosan dressing’s antibacterial capabilities by incorporating various agents such as AgSD [48], chlorhexidine diacetate [63] and minocycline hydrochloride [2] have been reported. 3.3 The Relevant Antibacterial Agents Silver ions, which are the most commonly used topical antimicrobial agent in burn wound care products, do not discriminate between cells involved in the healing process and pathogenic bacteria. Several recent tissue culture studies have shown that silver ions can cause lethal damage to both keratinocytes and fibroblasts [7, 53, 55, 57]. Such tests are probably a severe estimate, since rapid inactivation by chloride and protein occur in the wound, in the clinical environment [57]. However, the inactivation of silver ions concomitantly reduces their antibacterial potency. Since the bacterial and cellular toxic silver dose is within a similar range (7–55 mg/mL) [57], it has been suggested that silverbased products should be used with caution in situations where rapidly proliferating cells may be harmed, such as in superficial burns and the application of cultured cells [55, 57]. Despite growing evidence of bacterial resistance, antibiotics still represent an effective and selective treatment option against bacterial infections. The incorporation of broad-spectrum antibiotics such as gentamicin, ceftazidime or mafenide acetate in wound dressings can help reduce the bio-burden in the wound bed and thus prevent infection and accelerate wound healing. Aminoglycosides such as gentamicin are often used as prophylaxis when skin excisions and transplantation are undertaken. However, aminoglycosides have a narrow therapeutic index and are known for their potential nephro- and ototoxicity when used systemically, such that frequent drug and renal function monitoring are mandatory [79]. Ceftazidime is a third generation cephalosporin antibiotic that is very active against Gramnegative bacteria, including P. aeruginosa, and is a suitable antibiotic for the prophylaxis and therapy of bacterial infections in patients with severe burns [73, 79]. Mafenide is particularly appropriate in cases of burn wounds, since it 14 J. J. Elsner et al. Table 2 Physicochemical properties of the antibiotics used in the study [6] Antibiotic agent Molecular Water Antibacterial spectrum weight (g/ solubility mol) (mg/mL) Gentamicin 477.6 sulphate Ceftazidime 546.6 pentahydrate Mafenide acetate 246.3 100 5 250 Effective against a broad spectrum of Grampositive and Gram-negative bacteria A third-generation cephalosporin which displays a broad spectrum activity against Grampositive and Gram-negative bacteria. Unlike most third-generation agents, it is active against Pseudomonas aeruginosa Bacteriostatic for many Gram-negative and Gram-positive organisms, including Pseudomonas aeruginosa and certain strains of anaerobes. Mafenide is highly soluble and diffuses into and through eschar producing a marked reduction in the number of bacteria present, even in avascular tissue of secondand third-degree burns exhibits excellent antimicrobial activity and the best eschar penetration of any antibacterial agent [72]. However, mafenide causes severe side effects, especially when applied to large areas [52]. The controlled release of this antibiotic, alone or in combination with the other antibiotics reported in this study, may help prevent the occurrence of complications associated with conventional topical antibiotic treatment. The physicochemical properties of three these water soluble antibiotics are brought in Table 2. Their release directly to the wound, and in a controlled manner, should enable reaching a high local concentration while avoiding systemic toxicity, and therefore we chose to use them in our study which is presented in the following sub-chapter. 4 Novel Composite Antibiotic-Eluting Wound Dressings The previous section shows that there is currently no available synthetic dressing that combines the advantages of occlusive dressings with biodegradability and intrinsic topical antibiotic treatment. In order to obtain this combination of properties we have recently developed and studied a composite wound dressing based on the concept of core/shell (matrix) composite structures. Its characteristics are described here. Composites are made up of individual materials, matrix and reinforcement. The matrix component supports the reinforcement material by maintaining its relative positions and the reinforcement material imparts its special mechanical properties to enhance the matrix properties. Taken together, both materials synergistically produce properties unavailable in the individual constituent materials, allowing the designer to choose an optimum combination. In our application, a reinforcing Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 15 polyglyconate mesh affords the necessary mechanical strength to the dressing, while the porous Poly (DL-lactic-co-glycolic acid) (PDLGA) binding matrix is aimed to provide adequate moisture control and release of antibiotics in order to protect the wound bed from infection and promote healing. Both structural constituents are biodegradable, thus enabling easy removal of the wound dressing from the wound surface once it has fulfilled its role. This new structural concept in the field of wound healing is presented in Fig. 2. The freeze-drying of inverted emulsions technique which was used to create the porous binding matrix is unique in its ability to preserve the liquid structure in the solid state [85]. The viscous emulsion, consisting of a continuous PDLGA/chloroform solution phase and a dispersed aqueous drug solution, formed good contact with the mesh during the dip-coating process. Consequently, an unbroken solid porous matrix was deposited by the emulsion following freeze-drying (Fig. 2b). The freeze-drying of inverted emulsions technique has several advantages. First, it enables attaining a thin uninterrupted barrier, which unlike mesh or gauze alone can better protect the wound bed against environmental threats and dehydration. Second, it entails very mild processing conditions which enable the incorporation of sensitive bioactive agents such as antibiotics [15, 87] and even growth factors [85] to help reduce the bio-burden in the wound bed and accelerate wound healing. Fig. 2 The structure of the biodegradable composite wound dressing composed of polyglyconate fibers surrounded by a continuous PDLGA porous matrix: a Photograph of the wound dressing, b cross-sectional cryo-fractured SEM image showing two fibers and the binding matrix between them, c, d the microstructure of the porous matrix [18] 16 J. J. Elsner et al. Third, the microstructure of the freeze-dried matrix can be customized through modifications of the emulsion’s formulation to exhibit different attributes, namely different porosities or drug release profiles. Such structuring effects are described in this chapter. The mechanical and physical properties of these new wound dressings and their biological performance are also presented. Finally, a guinea pig model was used to evaluate the effectiveness of these antibiotic-eluting dressings and the main conclusions are brought here. 4.1 Structure-Controlled Release Effects The controlled release of antibiotics from wound dressings is challenging, since various related design considerations need to be addressed. Specifically, porosity which is desired to provide adequate gaseous exchange and absorption of wound exudates [48] may act as a two-edged sword; allowing rapid water penetration which typically leads to a rapid release of the water soluble active agent within several hours to several days [40, 71]. Structural effects on the controlled release of gentamicin and ceftazidime from our composite structures were extensively studied [15, 17] and the most important results are presented here. The emulsion’s formulation parameters which determine the porous matrix structure and also the resulting properties are the organic:aqueous (O:A) phase ratio, the drug content in the aqueous phase, the polymer content in the organic phase, the polymer’s initial molecular weight (MW) and also surfactants incorporated in the emulsion so as to increase its stability. The characteristic features of our studied samples are presented in Table 3. The basic formulations were used for the microstructure-release profile study. A highly interconnected porous structure poses almost no restriction to outward drug diffusion once water penetrates the matrix, and drug release in this case is most probably governed by the rate of water penetration into the matrix. Hence, the antibiotic release from our reference formulation (formulation 1, Fig. 3a, open circle) clearly demonstrates the prominent effect of pore connectivity on the burst release of the antibiotics, i.e. release of drug within the first 6 h. Samples with relatively low emulsion’s O:A phase ratio (up to 8:1) typically demonstrate much pore connectivity (Fig. 3b) and their in vitro release patterns display a burst release of approximately 95% (Fig. 3a, open circle). In contradistinction, porous shell structures derived from higher O:A phase ratios (for example 12:1), display reduced pore connectivity and a lower pore fraction (Fig. 3c and Table 3), resulting in a significant half-fold decrease in the burst release of antibiotics to approximately 45% (Fig. 3a, open triangle). An increase in the polymer’s MW from 100 to 240 kDa resulted in a tremendous effect on the shell microstructure. The porosity of the shell in this case was reduced to only 16% (Fig. 3d and Table 3). Since high viscosity increases the shear forces during the process of emulsification and also reduces the tendency of droplets to move, it is expressed in a significantly smaller pores and relatively thick polymeric domain between them. These changes in microstructure reduced Basic formulations Formulations with surfactants * 1. Reference 2. High O:A 3. High Polymer Content 4. High Polymer MW 5. BSA1: Ref, stabilized with BSA 6. BSA2: High O:A, stabilized with BSA 7. SPAN: High O:A, stabilized with Span Freeze-dried emulsion Porosity (%) Pore diameter (lm) 6:1 12:1 5 6:1 5 15 15 20 100 100 100 None None None 68 45 22 1.5 ± 0.6 1.6 ± 0.4 1.2 ± 0.9 6:1 6:1 5 5 15 15 240 83 16 63 0.5 ± 0.4 1.4 ± 0.3 12:1 5 15 83 35 1.4 ± 0.3 12:1 5 15 83 None BSA (1% w/v in the aqueous phase) BSA (1% w/v in the aqueous phase) Span80 (1% w/v in the organic phase) 45 1.1 ± 0.3 Relative to the polymer weight Relative to the liquid phase volume (organic or aqueous) ** Polymer Surfactant** MW (kDa) Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications Table 3 Structural characteristics of the ceftazidime-loaded porous matrix [16] Formulation O:A Drug loading* Polymer content in the (w/w) (%) organic phase**(w/v) (%) 17 18 J. J. Elsner et al. Fig. 3 a Controlled release of the antibiotic drug ceftazidime from composite structures based on various formulations. open circle—the reference formulation (formulation 1): 5% w/w ceftazidime and 15% w/v polymer (75/25 PDLGA, MW = 100 kDa), O:A = 6:1, open triangle—formulation 2: increased O:A phase ratio (12:1), open square—formulation 3: increased polymer MW (240 kDa), and open diamond—formulation 4: increased polymer content in the organic phase (20%). b–e SEM fractographs showing the effect of a change in the emulsion’s formulation parameters on the microstructure of the binding matrix for formulations 1–4, respectively [16] Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 19 the burst release of the encapsulated antibiotics to approximately 30% and enabled a continuous moderate release over a period of 1 month (Fig. 3a, open square). Finally, an increase in the emulsion’s polymer content to 20% w/v also resulted in a dramatic decrease in the burst release (Fig. 3a, open diamond). A higher polymer content in the organic phase results in denser polymer walls between pores after freeze-drying (Fig. 3e) and therefore poses better constraint on the release of drugs out of pores. Interestingly, samples containing a 20% polymer content exhibited a three-phase release pattern: an initial burst release, a continuous release at a declining rate during the first 2 weeks until release of 50% of the encapsulated drug, followed by a third phase of release of a similar nature reaching 99% release after 42 days. The second phase of release is governed by diffusion, whereas the third phase is probably governed by degradation of the host polymer which enables trapped drug molecules to diffuse out through newly formed elution paths. In other cases described thus far, drug release was governed primarily by diffusion, since almost the entire amount of drug was released before polymer degradation would in fact be able to affect the release profile. Thus, when drug diffusion out of the shell is restricted as in the case of high polymer content, and a considerable amount of drug still remains within the porous matrix, polymer degradation will contribute to further release the antibiotics, which leads to an additional release phase. Other modifications to the emulsion formulation included the addition of surfactants. Surfactants promote stabilization of the emulsion by reduction of interfacial tension between the organic and aqueous phases, resulting in refinement of the microstructure. We examined three matrix formulations loaded with surfactants (listed in Table 3), which display distinctly different micro-structural features (Fig. 4a–c and Table 3). The effect of the O:A phase ratio was examined on formulations containing bovine serum albumin (BSA) as surfactant. As expected, a higher O:A phase ratio, i.e., lower aqueous phase quantity, resulted in a smaller porosity of the solid structure. However, both microstructures were homogenous and characterized by a similar average pore size. The stabilization effect of Span 80 was even higher than that obtained using BSA, and therefore resulted in a smaller pore size (Table 3). The release profile of antibiotics from wound dressings varied considerably with the changes in formulation (Fig. 4d). Ceftazidime release from the dressings based on the BSA1 formulation was relatively short, reaching almost complete release of the encapsulated drug within 24 h. An increase in the emulsion’s O:A phase ratio from 6:1 to 12:1 reduced the burst release. Specifically, burst release values of 97 and 57% were recorded after 6 h for formulations BSA1 and BSA2, respectively, after which the release of the antibiotics from BSA2 dressings continued for 5 days at a decreasing rate. The ceftazidime release profile from the SPAN formulation was totally different. It exhibited a low burst release of 6% during the first 6 h of incubation and then a release pattern of a nearly constant rate for 10 days. Surfactant incorporation can contribute to the achievement of more than merely a stabilizing effect, by binding to antibiotics and thus counteracting drug depletion. We have found, for instance, that dressings containing mafenide in combination with albumin as surfactant display a lower burst release and a moderate release rate [15]. 20 J. J. Elsner et al. Fig. 4 a–c SEM fractographs demonstrating the microstructure of wound dressings based on formulations BSA1 (filled circle), BSA2 (light shaded filled square) and SPAN (filled triangle), respectively. d The controlled release of the antibiotic drug ceftazidime and e water loss, corresponding to each sample, together with water loss from a dense (non-porous) PDLGA (50/50, MW 100 kDa) film (dark shaded filled square) and from an uncovered surface (open square) [16] In summary, we demonstrated the release of antibiotic contents at high ([90%), intermediate (40–60%) and low (*5%) burst release rates and release spans ranging from several days to three weeks. The versatility of the drug release profiles was obtained through the effects of the inverted emulsion’s formulation parameters on the porous structure. In particular, lower burst release rates and longer elution durations can be achieved through structuring towards a reduced pore size, pore connectivity and total porosity. 4.2 Physical and Mechanical Properties 4.2.1 Moisture Management Successful wound healing requires a moist environment. Two parameters must therefore be determined: the water uptake ability of the dressing and the water Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 21 vapor transmission rate (WVTR) through the dressing. An excessive WVTR may lead to wound dehydration and adherence of the dressing to the wound bed, whereas a low WVTR might lead to maceration of healthy surrounding tissue and buildup of a back pressure and pain to the patient. A low WVTR may also lead to leakage from the edges of the dressing which may result in dehydration and bacterial penetration [4, 60]. It has been claimed that a wound dressing should ideally possess a WVTR in the range of 2,000–2,500 g/m2/day, half of that of a granulating wound [60]. In practice; however, commercial dressings do not necessarily conform to this range, and have been shown to cover a larger spectrum of WVTR, ranging from 90 (DermiflexÒ, J&J [80] to 3,350 g/m2/day (BeschitinÒ, Unitika [48]). Clearly, the WVTR is related to the structural properties (thickness, porosity) of the dressing as well as to the chemical properties of the material from which it is made. In this part of the study, we examined the specific emulsion formulations that included surfactants (BSA1, BSA2, SPAN, see Table 3). These were chosen based on emulsion stability and resultant microstructure (Fig. 4a–c), and also on drug release profiles (Fig. 4d). Evaporative water loss through the various dressings (Fig. 4e) was linearly dependant on time (R2 [ 0.99 in all cases), resulting in a constant WVTR, between 480–3,452 g/m2/day, depending on the formulation (Table 4). These results demonstrate how the WVTR can be customized based on modifications of the porous matrix’s microstructure. The lowest value is similar to that reported for film type dressings (e.g. Tegaderm, 491 ± 44 g/m2/day) [42], while the highest value is similar to that of foam type dressings (e.g. Lyofoam, 3,052 ± 684 g/m2/day) [42]. Further investigation of O:A phase ratios between 6:1 and 12:1 with albumin may generate a WVTR specifically in the 2,000–2,500 g/m2/ day range. A WVTR of 2,641 ± 42 g/m2/day which was achieved for 12:1 O:A with the surfactant Span 80 (formulation 7) is close to this range and seems the most appropriate. Water uptake by the wound dressing may occur either as the result of water entry into accessible voids in the porous matrix structure (hydration effect), or as the polymer matrix material gradually uptakes water and swells (swelling effect). Our water uptake patterns for wound dressings based on formulations (5) and (6) demonstrated both these effects (Fig. 5). Both types of wound dressing demonstrated a 3-stage water uptake pattern. Examination of the water uptake process through temporal micro-structural changes in the polymeric matrix sheds light on these stages, as follows: Table 4 Water vapor transmission rates, calculated for various wound dressings [16] Dressing type WVTR (g/m2/day) Dense (nonporous) film BSA1 BSA2 SPAN Open surface 356 ± 106 3,452 ± 116 480 ± 69 2,641 ± 42 6,329 ± 765 22 J. J. Elsner et al. Fig. 5 a Water uptake by two dressing formulations containing: 5% w/w ceftazidime, 15% w/v polymer (50/50 PDLGA, MW 100 kDa), stabilized with 1% w/v BSA: (filled circle) BSA1 (O:A = 6:1) and (filled square) BSA2 (O:A = 12:1), b–e E-SEM fractographs of BSA1, recorded during the water uptake experiment at 0 (b), 2 (c), 4 (d) and 14 days (e). In addition, larger domains of (b) and (e) are presented [17] Stage 1. (governed by hydration): during this stage of water uptake a quick flux of water associated with hydration of the porous structures was measured within 6 h after immersion in PBS for both types of dressings, as demonstrated in Fig. 5a. Water content then plateaued at values of 65% for the BSA1 formulation with the higher porosity (63%) and 55% for the BSA2 formulation with the smaller porosity (35%). Stage 2. A small decrease in water content during days 2–4, probably due to gradual shrinkage of pore walls and reorganization of the porous matrix (Fig. 5b–d). Such changes could be provoked by a combined effect of the polymer’s glass transition temperature which is very close to the incubation temperature (37°C) in combination with a softening effect of water on the polymer. It has been shown, for instance, that amorphous electrospun PDLGA fibrous mats undergo drastic Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 23 shrinkage after 1 day of in vitro incubation due to the relaxation of extended amorphous chains [89]. A 26% decrease in the void fraction of the polymer matrix (Fig. 5e) evidently caused a reduction in the water volume contained within the matrix (*20%) during this phase. Stage 3. (governed by swelling): After the fourth day of immersion in the aqueous medium, water uptake increased gradually and similarly for both types of dressing over the duration of 3 weeks, ultimately reaching a two-fold increase. The process of swelling is dependent on the polymer’s water affinity. Since PDLGA is not as hydrophilic as hydrogels or natural polymers used in this application, swelling occurred slowly. It is believed that the swelling effect was enhanced over time as hydrophilic end groups became more abundant due to polymer degradation by hydrolysis. This stage is also characterized by gradual thickening of the polymer walls due to the increased water uptake and creation of larger voids in the matrix due to polymer degradation (Fig. 5e). The combination of these two changes, which occurred in parallel, resulted is a coarser microstructure. 4.2.2 Mechanical Properties The mechanical properties of a wound dressing are an important factor in its performance, whether it is to be used topically to protect cutaneous wounds or as an internal wound support, e.g. for surgical tissue defects or hernia repair. Furthermore, in the clinical setting, appropriate mechanical properties of dressing materials are needed to ensure that the dressing will not be damaged by handling. Porous structures typically possess inferior mechanical properties compared to dense structures, yet in wound healing applications porosity is an essential requirement for diffusion of gasses, nutrients, cell migration and tissue growth. Most wound dressings are therefore designed according to the bi-layer composite structure concept and consist of an upper dense ‘‘skin’’ layer to protect the wound mechanically and prevent bacterial penetration and a lower spongy layer designed to adsorb wound exudates and accommodate newly formed tissue. Our new dressing design integrates both structural/mechanical and functional components (e.g., drug release and moisture management) in a single composite layer [17]. It combines relatively high tensile strength and modulus together with good flexibility (elongation at break). It actually demonstrated better mechanical properties than most other dressings currently used or studied, as demonstrated in Table 5. The initial mechanical properties of natural polymers such as collagen or gelatin can be satisfactory. However, considerable degradation of these properties is expected to occur rapidly due to hydration [62] and enzymatic activity [43]. The results of the 3 weeks degradation study of our wound dressings show a significant decrease only in Young’s modulus (Fig. 6). The maximal stress and strain of our composite wound dressing (24 MPa and 55%, respectively) are dictated mainly by the mechanical properties of the reinforcing fibers which fail first during breakage. At these time periods they are not subjected to considerable degradation, which 24 J. J. Elsner et al. Table 5 Mechanical properties of various wound dressings [17] Material/format Elastic modulus (MPa) BSA1 (composite polyglyconate mesh, coated with PDLGA porous matrix) Electrospun poly-(L-lactide-co-e-caprolactone) (50:50) mat [45] Electrospun gelatin mat [45] Electrospun collagen mat [62] ResolutÒ LT regenerative membrane (Gore). Glycolide fiber mesh coated with an occlusive PDLGA membrane [49] KaltostatÒ (ConvaTec) Calcium/sodium alginate fleece [9] Tensile strength (MPa) Elongation at break (%) 126 ± 27 24.2 ± 4.5 55 ± 5 8.4 ± 0.9 4.7 ± 2.1 960 ± 220 490 ± 52 1.6 ± 0.6 11.4 ± 1.2 11.7 17.0 ± 4.4 0.9 ± 0.1 10.8 ± 0.4 1.3 ± 0.2 20 Fig. 6 a Tensile stress–strain curves for wound dressings immersed in water for 0 (–0–), 1 (–1–) 2 (–2–), and 3 (–3–), weeks, b Young’s modulus, c tensile strength and d maximal tensile strain as a function of immersion time. Comparison was made using ANOVA and significant differences are indicated (asterisk) [17] explains the constancy in these properties. In contradistinction, the Young’s modulus of the dressings is considerably affected by the properties of the binding matrix that makes up the largest part of the cross-sectional area. The degradation of the matrix material which is clearly in progress after 2 weeks of exposure to PBS (Fig. 5e) thus leads to a decrease in Young’s modulus. The mechanical properties of our wound dressings are superior to those reported before, and remain Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 25 good even after 3 weeks of degradation (Young’s modulus of 69 MPa, maximal stress 24 MPa and maximal strain 61%), as demonstrated in Fig. 6. It should also be mentioned that delamination of the fiber-matrix interface may affect the integrity of the wound dressing as well as barrier properties. We have therefore examined the effect of degradation on the interface and found that the dressing material maintained its integrity for the duration of at least 2 weeks exposure to aqueous medium, despite a gradual ongoing process of degradation of the matrix component. SEM observations (Fig. 7) showed high quality fibermatrix interface, i.e. contact between the two components still exists as degradation proceeds [17]. In summary, the mechanical properties of our wound-dressing structures were found to be superior, combining relatively high tensile strength and ductility, which changed only slightly during 3 weeks of incubation in an aqueous medium. The parameters of the inverted emulsion as well as the type of surfactant used for stabilizing the emulsion were found to affect the microstructure of the binding matrix and the resulting physical properties, i.e., water absorbance and water vapor transmission rate. 4.3 Biological Performance 4.3.1 Bacterial Inhibition The strategy of drug release to a wound depends on the condition of the wound. After the onset of an infection, it is crucial to immediately respond to the presence of large numbers of bacteria ([105 CFU/mL) which may already be present in the biofilm [30], and which may require antibiotic doses of up to 1,000 times those needed in suspension [12, 23]. Following the initial release, sustained release at an effective level over a period of time can prevent the occurrence of latent infection. We have shown that the proposed system can comply with these requirements (sect. 4.1). The time-dependent antimicrobial efficacy of these antibiotic-eluting wound dressing formulations was tested in vitro by the following two complementary methods: 1. The corrected zone of inhibition test (CZOI) [18], which is also termed the disc diffusion test. According to this method presence of bacterial inhibition in an area that exceeds the dressing material (CZOI [0) can be considered beneficial. This method gives a good representation of the clinical situation, where the dressing material is applied to the wound surface, allowing the drug to diffuse to the wound bed. The results from this method are dependent on the rate of diffusion of the active agent from the dressing, set against the growth rate of the bacterial species growing on the lawn, and are highly dependent on the physicochemical environment. 2. A release study from selected wound dressings in the presence of bacteria was also performed, in order to study the effect of drug release on the kinetics of 26 J. J. Elsner et al. Fig. 7 SEM fractographs demonstrating the interface between the reinforcing fiber and porous matrix for specimens based on formulation BSA1, immersed in aqueous medium for: a start point, b 1 week, and c 2 weeks [17] residual bacteria [18]. This method, which is termed viable counts, provide valuable information on the kill rate, which is a key comparator for different formulations and physicochemical conditions. The bacterial strains S. aureus, Staphylococcus albus (S. albus) and P. aeruginosa were used in this study. The results for wound dressings stabilized with BSA are presented in Figs. 8, 9 and 10. Wound dressings containing gentamicin demonstrated excellent antimicrobial properties over 2 weeks, with Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 27 Fig. 8 The inhibition of P. aeruginosa, S. albus and S. aureus growth around wound dressings based on the emulsion formulations containing 10% gentamicin or 10% ceftazidime and stabilized with 1% BSA, PBS incubation times prior to the test are indicated next to each sample [18] bacterial inhibition zones extending well beyond the dressing margin at most times (Figs. 8 and 9). Interestingly, inhibition zones around dressing materials containing gentamicin remained close to constant over time and for the different drug loads. The largest CZOI were measured for the Gram-positive bacteria (S. aureus. and S. albus) and especially for S.albus. Despite having the lowest minimal inhibitory concentration (MIC) (Table 6), The Gram-negative P. aeruginosa was least inhibited, and exhibited the smallest CZOI (Figs. 8 and 9). This was not the case for ceftazidime-loaded materials, for which CZOI were found to decrease over time, and with lower drug loads. In contradistinction to gentamicinloaded materials, ceftazidime was found to be most effective against P. aeruginosa and less effective against S. albus and S. aureus, and in good correlation with their MIC’s (Table 6). The CZOI was also evaluated for dressing materials with a reduced burst release, stabilized by Span, focusing on the mid-range 10% antibiotic loading ratio. Samples containing gentamicin exhibited similar inhibition zones to those shown when BSA was used as surfactant. In contradistinction, both the magnitude and the duration of the inhibition effect of ceftazidime-loaded materials were reduced [18]. 28 J. J. Elsner et al. Fig. 9 Histograms showing the effect of drug release on corrected zone of inhibition (CZOI) around (1% w/v) BSA loaded wound dressings (n = 3) containing 5% (w/w) (filled square), 10% (w/w) ( ) and 15% (w/w) (open square) drug, as a function of pre-incubation time in PBS: a–c—gentamicin-loaded wound dressings, d–f—ceftazidime-loaded dressings. The bacterial strain (P. aeruginosa, S. albus and S. aureus) is indicated [18] Table 6 Minimum inhibitory concentrations of antibiotics [18] Microorganism MIC (lg/mL) Pseudomonas aeruginosa Staphylococcus albus Staphylococcus aureus Gentamicin Ceftazidime 2.5 3 6.3 6.3 12.5 12.5 Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 29 Fig. 10 Number of colony forming units (CFU) versus time, when initial concentrations of 107– 108 CFU/mL were used: a P. aeruginosa, b S. albus and c S. aureus. The releasing wound dressing discs (D = 10 mm) were derived from 1% (w/v) BSAstabilized emulsions containing 10% (w/w) gentamicin (filled square) or 10% (w/w) ceftazidime (filled circle). Bacteria in the presence of PBS only served as control (asterisk) [18] The purpose of the viable counts experiments was to monitor the effectiveness of cumulative antibiotic release from the wound dressings in terms of the residual bacteria compared to initial bacterial inoculations of 107–108 CFU/mL which correspond to severe infection. This investigation focused on samples based on 30 J. J. Elsner et al. BSA-stabilized emulsions containing 10% antibiotics. Bacteria present in PBS only served as the control. Curves describing the decrease in the number of bacteria due to antibiotic release also demonstrate the superiority of gentamicinloaded dressing materials over ceftazidime (Fig. 10). High bacterial inoculations of 107–108 CFU/mL were decreased by 99.99% after 1 (P. aeruginosa and S. albus) to 3 days (S. aureus) in the presence of the gentamicin-loaded wound dressing material. Under similar conditions, the ceftazidime-loaded dressing material demonstrated a 99% decrease in P. aeruginosa and S. albus only after 3 days, and its effect on S. aureus was even lower. 4.3.2 Cell Cytotoxicity In order to complete the results of bacterial inhibition, it is also necessary to ensure that the dressing material we developed is not toxic to the cells that participate in the healing process. Previous studies have shown that dressing materials may impose a toxic effect on cells, caused by the dressing material itself, its processing, or due to the incorporation of antimicrobials [13, 55]. We assessed cell viability by observations of cell morphology, and by use of the Alamar-Blue assay, which is comparable to the MTT assay in measuring changes in cellular metabolic activity [29]. This method involves the addition of a non-toxic fluorogenic redox indicator to the culture medium. The oxidized form of AB has a dark blue colour and little intrinsic fluorescence. When taken up by cells, the dye becomes reduced and turns red. This reduced form of AB is highly fluorescent. The extent of the AB conversion, which is a reflection of cell viability, can be quantified spectrophotometrically at wavelengths of 570 and 600 nm. The AB assay is advantageous in that it does not necessitate killing the cells (as in the MTT assay), thus enabling day by day monitoring of the cell cultures. The AB assay was performed on human fibriblast cell cultures before introducing the dressing materials and then every 24 h for 3 days. We saw no difference in the appearance of the cell cultures over the 3 days during which they were exposed to the dressing material devoid of antibiotics. The AB assay also shows a stable preservation of cellular viability. Thus, we are assured that the dressing material itself and its processing by freeze-drying of inverted emulsions do not inflict a toxic effect. Similar results were obtained for all the dressing materials containing antibiotics. No more than a 10% reduction in the metabolic activity of cell cultures was measured and in most cases metabolic activity even increased as the cells became more confluent (Fig. 11). These results are promising, when compared to studies reporting the similar testing of commonly used silver-based dressing materials. Burd et al. [7] and Paddle-Leinek et al. [55] have reported that such dressings induce a mild to severe cytotoxic effect on keratinicytes and fibroblasts grown in culture, which correlated with the silver released to the culture medium. Specifically, it was shown that commercial TM dressings such as Acticoat , AquacelÒ Ag and ContreetÒ Ag reduce fibroblast viability in culture by 70% or more [55]. All silver dressings were shown to delay Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 31 Fig. 11 Histograms demonstrating changes in the viability of dermal fibroblast cultures (Alamar Blue assay) in the presence of wound dressing discs (D = 10 mm): a BSA-stabilized wound dressings (n = 3) containing 5 or 15% (w/w) gentamicin, b BSA and Span stabilized wound dressings containing 5 or 15% ceftazidime. Dressing materials devoid of antibiotics and pristine cell cultures served as control [18] wound reepithelialization in an explant culture model, and AquacelÒ Ag and ContreetÒ Ag were found to significantly delay reepithelialization in a mouse excisional wound model [7]. These findings emphasize the superiority of the proposed new antibiotic-eluting wound dressings over dressings loaded with silver ions. 32 J. J. Elsner et al. In summary, both types of microbiological studies showed that the investigated antibiotic-eluting wound dressings are highly effective against the three relevant bacterial strains. Despite severe toxicity to bacteria, the dressing material was not found to have a toxic effect on cultured fibroblasts, indicating that the new antibiotic-eluting wound dressings represent an effective and selective treatment option against bacterial infection. 4.4 In vivo Study The guinea pig is often used as a dermatological and infection model [3, 36, 54, 83]. Research on guinea pigs has included topical antibiotic treatment [5], delivery of delayed-release antibiotics [22], and investigation of wound dressing materials [37, 47]. A deep partial skin thickness burn is an excellent wound model for the evaluation of wound healing, not only for contraction and epithelialization of the peripheral area such as in third degree burns, but also for evaluation of the recovery of skin appendages, to serve as the main source for the re-epithelization, which completes the healing process. The metabolic response to severe burn injury in guinea pigs is very similar to that of the human post-burn metabolic response [31]. Furthermore, bacterial colonization and changes within the complement component of the immune system in human burn victims is analogous to guinea pigs affected by severe burns [3]. Such a model was therefore used in the current study to evaluate the effectiveness of our novel composite antibiotic-eluting wound dressing. Our in vivo evaluation of the antibiotic-eluting wound dressings in a contaminated wound demonstrated its ability to accelerate wound healing compared to an unloaded format of the wound dressing and a non-adherent dressing material (MelolinÒ). Faster epithelialization of the wound was measured in both release strategies, fast antibiotic release (such as in Fig. 4d, filled square) and slow antibiotic release (such as in Fig. 4d, filled triangle), but was significantly better for animals treated with the slow release rate. The results are described in detail elsewhere [19 (submitted)]. From a practical aspect, faster epithelialization leads to less pain to the patient, shorter hospitalization, a better healing quality. The new current dressing material shows promising results. It does not require bandage changes and offers a potentially valuable and economic approach for treating the life-threatening complication of burn-related infections. 5 Conclusion This chapter presents an overview of wound dressings with controlled release of antibacterial agents. These include wound dressings based on both types of polymers, synthetic and natural. A special part of this chapter focused on our novel biodegradable occlusive wound dressings, based on a polyglyconate mesh and a Novel Composite Antibiotic-Eluting Structures for Wound Healing Applications 33 porous PDLGA binding matrix. These composite dressings were prepared by dipcoating woven meshes in inverted emulsions, followed by their freeze-drying. Their in vitro investigation focused on the microstructure, mechanical and physical properties and the release profiles of the antibiotic drugs and their effects on bacterial inhibition. The main results were brought here, with emphasis on engineering aspects related to wound dressings. Our new composite structures demonstrated combination of good mechanical properties with desired physical properties and adjustable controlled release of the antibiotic drugs from the binding matrix, which resulted in impressive bacterial inhibition effect and biocompatibility. Hence, these new wound dressings are potentially very useful as burn and ulcer dressings with enhanced protection against infection and reduce the need for frequent dressing changing. Furthermore, we have shown that modifications in the emulsion formulation enables adapting the desired properties to the wound characteristics, and may thus enhance wound healing. New designs based on structured wound dressings may thus advance the therapeutic field of wound healing. Acknowledgments The authors are grateful to the Israel Science Foundation (ISF, grant no. 1312/07) and to the Israel Ministry of Health (grant no. 3-3943) for supporting this research. References 1. Altoparlak, U., Erol, S., Akcay, M.N., Celebi, F., Kadanali, A.: The time-related changes of antimicrobial resistance patterns and predominant bacterial profiles of burn wounds and body flora of burned patients. Burns 30(7), 660–664 (2004) 2. Aoyagi, S., Onishi, H., Machida, Y.: Novel chitosan wound dressing loaded with minocycline for the treatment of severe burn wounds. Int. J. Pharm. 330, 138–145 (2007) 3. Bjornson, A.B., Bjornson, H.S., Lincoln, N.A., Altemeier, W.A.: Relative roles of burn injury, wound colonization, and wound infection in induction of alterations of complement function in a guinea pig model of burn injury. J. Trauma 24, 106–115 (1984) 4. Boateng, J.S., Matthews, K.H., Stevens, H.N., Eccleston, G.M.: Wound healing dressings and drug delivery systems: a review. J. 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