Functional electrospun fibers for the treatment of human skin wounds

Accepted Manuscript Functional electrospun fibers for the treatment of human skin wounds Jing Wang, Maike Windbergs PII: DOI: Reference:

S0939-6411(17)30628-8 http://dx.doi.org/10.1016/j.ejpb.2017.07.001 EJPB 12557

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

19 May 2017 3 July 2017 4 July 2017

Please cite this article as: J. Wang, M. Windbergs, Functional electrospun fibers for the treatment of human skin wounds, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: http://dx.doi.org/10.1016/j.ejpb. 2017.07.001

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Functional electrospun fibers for the treatment of human skin wounds Jing Wang1 and Maike Windbergs1*

1

Institute of Pharmaceutical Technology and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt am Main, Germany

Abstract Wounds are trauma induced defects of the human skin involving a multitude of endogenous biochemical events and cellular reactions of the immune system. The healing process is extremely complex and affected by the patient´s physiological conditions, potential implications like infectious pathogens and inflammation as well as external factors. Due to increasing incidence of chronic wounds and proceeding resistance of infection pathogens, there is a strong need for effective therapeutic wound care. In this context, electrospun fibers with diameters in the nano- to micrometer range gain increasing interest. While resembling the structure of the native human extracellular matrix, such fiber mats provide physical and mechanical protection (including protection against bacterial invasion). At the same time, the fibers allow for gas exchange and prevent occlusion of the wound bed, thus facilitating wound healing. In addition, drugs can be incorporated within such fiber mats and their release can be adjusted by the material and dimensions of the individual fibers. The review gives a comprehensive overview about the current state of electrospun fibers for therapeutic application on skin wounds. Different materials as well as fabrication techniques are introduced including approaches for incorporation of drugs into or drug attachment onto the fiber surface. Against the background of wound pathophysiology and established therapy approaches, the therapeutic potential of electrospun fiber systems is discussed. A specific focus is set on interactions of fibers with skin cells/tissue as well as wound pathogens and strategies to modify and control them as key aspects for developing effective wound therapeutics. Further, advantages and limitations of controlled drug delivery from fiber mats to skin wounds are discussed and a future perspective is provided.

Keywords: electrospinning; wound healing; drug delivery; surface modification; controlled release

1. The human skin, wounds and healing processes For a detailed understanding of wound healing processes, extensive knowledge about the structure and functions of human skin is inevitable. Functionally acting as the first-line biological barrier, the skin protects the human body against harmful environmental influences and regulates the body´s hydration state. The skin forms a multilayer structure composed of the main layers, epidermis and dermis, which are located above the subcutaneous tissue containing abundant fat. During continuous renewal of the skin, keratinocytes are formed in the basal part of the epidermis and gradually migrate towards the outer layers of the skin, finally forming the outermost sheet called stratum corneum composed of dead cells embedded into a lipid matrix. The second most occurring cell type is represented by fibroblasts residing in the dermis. Both cell types are embedded in a three-dimensional, tissue-forming scaffold, the so called extracellular matrix (ECM). The ECM is composed of thin fibers based on natural polymers like collagen, elastin and fibrinogen and provides a flexible and dynamically changing tissue environment. Further, secretion of signaling molecules and growth factors takes place in the ECM, playing an important role for tissue growth and regeneration. Upon injury of the skin, several signaling cascades are initiated complemented by reactions of different cell types including immune cells. The mode and extent of such reactions is naturally dependent on the course and severity of the injury. Upon injuries which affect deep dermis layers, damaged blood and lymphatic vessels leak fluid counteracted by hemostasis (Fig. 1A). Physiochemical and biological changes of the injured tissue trigger damage signals initiating the subsequent wound repair. Platelets start to aggregate in the wound bed and secrete multiple growth factors (like epidermal growth factor, platelet-derived growth factor etc.) formatting a clot to plug the defect area [1]. This clot serves as the provisional matrix filling in the lesion, while vasoconstriction additionally limits leakage of the vessels and tissue. Simultaneously, the inflammatory phase is initiated with the recruitment of inflammatory cells triggered by multiple chemokines from platelets (Fig. 1B) [2]. These inflammatory cells like polymorphonuclear cells and other cells such as keratinocytes release growth factors and cytokines to regulate signaling systems [3]. Macrophages and other immune cells are stimulated and migrate towards the wound to dispose of cell debris and fight invading bacteria. Angiogenesis occurs at this phase and new blood vessels transport essential nutrients to the wound bed [1]. Finally, re-epithelialization is initiated in the proliferation phase by keratinocytes proliferating and migrating across the wound site (Fig. 1C) [3]. Fibroblasts are activated and differentiated into myofibroblasts to produce substances and regulate other cells to grow and form granulation tissue. And the provisional ECM is degraded with new ECM generated mainly by (myo)fibroblasts [2]. The final maturation phase is governed by the rearrangement of the newly formed ECM with more type I collagen replacing type III collagen (Fig. 1D). The proportion of proteoglycans and water decreases, while elastin appears in ECM to increase the elasticity [2]. Further, collagen fibers rearrange their structures with increasing interfibrillar binding and diameter [4]. Fig. 1 Four wound healing phases

As described above, wound healing is a highly complex and dynamic process which requires several weeks to be completed. However, a variety of different factors could affect those processes leading to impaired wound healing, including local factors such as oxygenation, infection, foreign body reaction and venous insufficiency, and systemic factors such as age and gender, sex hormones, psychological stress, diabetes as well as inappropriate medication [5]. If there is no healing after 12 weeks, such wounds are considered as being chronic. A prominent example for a chronic wound is the foot ulcer, which is one of the most common complications associated with diabetes mellitus; often additionally affected by infection pathogens. Due to the steadily aging society and the increasing incidence of wound infections and chronicity, effective therapy of wounds is a major clinical need. 2. Therapy of wounds Therapeutic wound care dates back to ancient Sumerians and Egyptians who made use of natural products such as mud, milk, plants, honey and animal fats. Since then, the therapeutic approaches have drastically changed. Today, the basic strategy relies on preventing, emerging or eradicating existing infections in combination with accelerating the healing process with structural and functional restoration of the skin [6]. For this purpose, the wounded tissue is firstly cleaned, treated with antiinfectives or antiseptics and physically covered with a wound dressing. State-of-the-art therapeutics mostly comprise liquid (such as solutions, emulsions and suspensions) or semi-solid (such as ointments, creams and foams) formulations. Despite their convenient application to the wound site, such formulations can only provide a short-term therapy as drug release is rather fast. Further, exudate from oozing wounds is prone to wash off the actives. For semi-solid systems, the excipients might even disturb wound healing or limit gas exchange. Recently, based on a better understanding of wound pathophysiology and wound healing processes, the established “dry” healing environment was replaced by a “moist” wound healing atmosphere, which could prevent tissue dehydration, cell death and further promote faster wound healing through angiogenesis acceleration and improve cellular communication [7]. This change of paradigms also resulted in a strong demand for suitable wound dressings. Such systems should provide: I) physical

protection against external trauma; II) inhibition of bacterial invading; III) wound exudate absorption; IV) maintenance of physiological temperature and moist environment; V) free gas and fluid exchange; VI) mechanical flexibility and easy removal without adhesion; VII) good biocompatibility [8]. A variety of wound dressings is currently available on the market, ranging from basic dry dressings to more sophisticated ones like hydrogel dressings, chemical-impregnated dressings etc. Dry dressings composed of gauze pads can protect wound tissue against mechanical trauma, and they are widely used due to their simple application and inexpensive price. However, their application is limited to wounds with small amounts of exudate. Additional tissue damage might occur upon removal of dressings with adhesive edges. Hydrogel dressings can absorb wound exudate, create a moist environment and do not adhere to the wound tissue. In many cases, additional antibiotic treatment is needed for infected wounds. For this purpose, dressings can be impregnated with active pharmaceutical ingredients (such as antibiotics, silver, collagen, povidone-iodine) intended to eradicate infections and accelerate healing process. However, release of such actives is not controlled over the intended therapeutic time interval and thus requires frequent exchange of impregnated dressings [9-12]. On the search for advanced wound therapeutics, electrospun fibers currently gain considerable interest as multifunctional wound dressings. Depending on the material and the dimensions, such fiber mats simulate the structure of the native ECM (Fig. 2). Further, such mats provide mechanical protection while at the same time preventing invasion of bacteria, but allowing for gas exchange and moist wound environment. Depending on the material, excess wound exudate can be absorbed and the fibers can be functionalized with active ligands. In addition, such fibers can be loaded with actives which can be released with controlled kinetics to allow for appropriate therapeutic dosing. Fig. 2 Electron micrographs of electrospun fibers and native human ECM.

3. Electrospun fibers for wound application 3.1 The principle of electrospinning As already mentioned above, electrospinning is a versatile approach to produce fine fibers with diameters in the nano- and micrometer range from polymer solutions or melts using electrostatic forces. The basic equipment is composed of three parts, a syringe with a metal needle for conveying the polymer solution with a controlled feeding rate, a high voltage generator to charge the conveyed liquid as well as a metal collector for deposition of the final fibers [13]. In a stable electrospinning process, the high voltage generates mutual electrical repulsive forces in the liquid to overcome its surface tension at the tip of the needle. A liquid jet is formed which is directed towards the metal collector which is also attached to the voltage generator. Before deposition of the solid fibers on the collector, the solvents are evaporated on the way from the needle to the collector, while in case of melts, the fibers solidify via heat dissipation (Fig. 3). The process is complex and numerous factors can influence its outcome. On the one hand, this is advantageous for a platform technology, as fiber morphology, diameter, properties etc. can systematically be modified. On the other hand, each individual process has to be well understood to allow for its reproducibility. The influencing parameters can be categorized into solution parameters (also addressing melts), processing parameters

and environmental parameters [13]. The solution parameters comprise the properties of the polymers, solvents and additional excipients governing viscosity, conductivity and surface tension of the solution. In contrast, processing parameters consist of solution feeding rate, applied voltage and tip to collector distance, while environmental parameters include temperature and humidity. All of these parameters also additionally influence each other, so that each of them has to be well balanced to generate fibers with desired characteristics. Fig. 3 Electrospinning setup and photograph and SEM image of electrospun fibers

3.2 Incorporation of drugs into electrospun fibers Electrospun fibers provide multiple options for drug incorporation. Drug molecules can directly be embedded into the polymer fiber matrix or be attached to the fiber surface. This holds for small and rather robust molecules as well as for sensitive macromolecules like peptides and proteins. However, the choice of a suitable polymer-solvent system is challenging. For instance, while water-soluble and thus hydrophilic polymers are ideal for incorporation of proteins and peptides, their release from such systems is rather fast and contradictory to the mostly desired controlled release over longer time intervals. Incorporation into fibers based on rather hydrophobic polymers which resulting into controlled release kinetics requires organic solvents which are prone to affect the stability of the active, and consequently its pharmacological effect. To overcome these problems, different approaches have been developed for incorporation of actives into fibers [14]. The following paragraphs provide an overview about these approaches. 3.2.1 Blend electrospinning From a practical viewpoint, electrospinning of a mix (blend) containing polymer, active and solvent is advantageous, as it is a simple one-step procedure. For synthetic polyesters like polycaprolactone (PCL) or poly(lactic-co-glycolic acid) (PLGA), organic solvents like chloroform, dichloromethane or acetone are widely used, while for hydrophilic polymers like polyvinylalcohol (PVA), gelatin or chitosan mostly water is applied as the solvent. To optimize the physicochemical properties of the polymer solution like surface tension, conductivity and vapor pressure, mixtures of different solvents are employed [15]. For example, chloroform can be combined with methanol or ethanol for spinning of PCL. Ideally, the active and the polymer should homogeneously be distributed within the final electrospun fibers. However, during solvent evaporation drug molecules can be dragged along with the evaporation front in the solidifying fiber. These molecules would be deposited on the fiber surface and thus be easily released and contribute to an undesired burst release (Fig. 4A) [16-21].

3.2.2 Emulsion electrospinning To overcome the mentioned limitations of blend electrospinning, emulsion electrospinning was developed. For this purpose, an aqueous phase (mostly containing the active) and an organic phase (containing the polymer) are mixed to form an emulsion, which is subsequently spun. The resulting fibers either conserve the emulsion structure with localized regions of the inner phase in the outer phase forming the fibers, or even more likely, the initial emulsion phases form a core-shell structure in the final fibers with the inner phase of the emulsion coalescing during fiber formation to form the core of the fibers (Fig. 4B) [22]. Emulsion electrospinning has successfully been used for encapsulation of proteins into biodegradable polymers. However, potential limitations include uncontrolled protein denaturation at the organic solvent interface and harsh conditions during initial emulsification [23]. For instance, Yang et al. [24] embedded basic fibroblast growth factor (bFGF) into poly(ethylene glycol)-poly(DL-lactide) (PELA) fibers via emulsion electrospinning. A core-shell structure was obtained with a low burst release of 14% ± 2.2%, followed by a gradual release over 4 weeks. The same group also encapsulated bFGF-encoding plasmid into PELA fibers, and got a similar sustained release profile [25]. 3.2.3 Co-axial electrospinning For direct generation of core-shell fibers, co-axial electrospinning is applied. For this purpose, two kinds of solutions are simultaneously fed through a nozzle with two concentric openings. Ideally, the two liquids would form a stable core-shell jet with one liquid in the core and one liquid as a sheet flow (Fig. 4C) [23]. Even more challenging, both liquids have to solidify in the same time interval before depositing on the collector. Compared to emulsion electrospinning, protein denaturation can be avoided through separately dissolving the protein in the core solution. This technique has widely been used to encapsulate fragile macromolecules for controlled release over a longer time period. Since two concentric nozzles are applied in coaxial electrospinning with two separate polymer systems, the influencing parameters on such a process are much more complex and a further understanding and standardization of this technique is demanded [23, 26]. Jin et al. [27] encapsulated multiple epidermal induction factors (EIF) including insulin, epidermal growth factor (EGF), hydrocortisone and retinoic acid with poly(L-lactic acid)-co-poly-(ɛcaprolactone) (PLLCL) and gelatin solutions through blend or co-axial electrospinning. An initial burst release of EGF with 44.9% from EIF bending nanofibers was detected within first three days, followed by a sustained release over 15 days by 77.8%. No burst release of EGF was obtained from EIF encapsulated nanofibers produced by co-axial electrospinning, with a more stable and sustained release of 50.9% after 15 days. 3.2.4 Surface immobilization Besides incorporating the active molecules into fibers during the electrospinning process, they can also be absorbed onto the surface of the fibers through physical or chemical interactions. This method often requires modification of the fiber forming polymers with special ligands like active amine groups (Fig. 4D). For example, 1,6-diaminohexane was used to couple an amine group to PCL, PCL/collagen or PCL/gelatin nanofibers, followed by activation of the amine end groups via suberic acid bis(Nhydroxy-succinimide ester) (NHS), finally the active was covalently conjugated to the polymer [28, 29]. Pegg et al. [30] prepared ammonium alginate-derived nanofibers with the potential to carry diverse cargos like lidocaine, neomycin and papain for wound healing. Besides, PEG damine was combined with PCL to constitute a copolymer with amine groups [31], while polydopamine as an adhesive bridge was used to couple growth factors onto PLGA fibers [32, 33]. Besides polypeptides, silver nanoparticles were also loaded onto nanofibers through post immobilization. Rivero et al. [34] prepared poly(acrylic acid) nanofibers, followed by loading of Ag+ onto fibers through ion exchange

by available carboxylic groups of poly(acrylic acid), finally silver nanoparticles were obtained via reduction by 0.1 M dimethylamine borane solution. The concept of post-spinning surface immobilization offers numbers of possibilities for localization and sustained release of actives. But this technique has been limited by the numerous steps of chemical reactions, and it requires electrospun fibers with high stability in aqueous solution where such reactions take place. Fig.4 Strategies for drug incorporation

4. Polymers for electrospinning A broad variety of different polymers can be utilized for fabrication of electrospun fibers for biomedical applications ranging from synthetic substances up to natural polymers from different sources. The most prominent examples are presented in the next few paragraphs. 4.1 Synthetic polymers Poly(ε-caprolactone) or PCL is a semicrystalline and biodegradable polyester. Under physiological conditions, the ester linkages undergo hydrolysis and degrade into nontoxic products. In an in vivo degradation study by Sun et al. [35], capsules prepared using PCL of 66 kDa could retain their intact shape after 24 months’ subcutaneous implantation, and degraded into smaller fragments of 8 kDa in 30 months. These properties make PCL a promising material for long-term implantable devices. Despite the reported biocompatibility of PCL, there are also studies showing material remaining within the recovery tissue may lead to inflammation and foreign body reactions [36]. However, PCL is still a widely used polymer for electrospinning due to its favorable mechanical characteristics. And fibers are easily obtained through either spinning pure PCL [37-41], or combined with natural hydrophilic polymers, for example, collagen [42], gelatin [43] or chitosan [44]. Poly(lactic-co-glycolic acid) or PLGA is a copolymer based on varying ratios of polylactic acid (PLA) and polyglycolic acid (PLG). Based on the ratio of the two components and the degree of polymerization, PLGA has a tunable degradation profile from weeks to years. In general, higher lactide content and higher molecular weight result in a slower degradation of the copolymer. In addition, an acid end group leads to a faster degradation. In general, degradation takes place by hydrolysis of ester linkages, with water and carbon dioxide as the final end products. Pure PLGA

fibers have successfully been prepared. However, shrinkage of fibers has been reported in physiological conditions [17, 45-48]. Poly(vinyl alcohol) or PVA is a water soluble polymer, resulting in a rapid disintegration of PVA fibers in aqueous solution [49]. Thus, for wounds with exudate or controlled release of incorporated drugs, such fibers need further modification to retain their structural stability. Pelipenko et al. [50] compared the structure stability of PVA nanofiber matrices by applying three post-treatment methods: methanol, ethanol and high temperature. Nanofiber matrices under thermal stabilization could retain their structure for 96 hours. Besides, cross-linking with saturated glutaraldehyde vapors was also applied to stabilize PVA based fibers, which introduced a smaller porosity and reduced tensile strength [51]. Other synthetic polymers like polyvinylpyrrolidone or PVP [52], polyurethane or PU [53], biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV [54-56], polyethersulfone or PES [57], polyvinylidene fluoride or PVDF [53] and polyethylene oxide or PEO [58] are also widely used to fabricate nanofiber matrices for wound applications. 4.2 Natural polymers Chitosan is a biodegradable polysaccharide produced by deacetylation of chitin, a naturally occurring and abundantly available polymer. Chitosan has a positive charge owning to the protonation of amino groups, and it is soluble in water under acidic pH, and insoluble in neutral or basic conditions. Chitosan and its derivatives are favorable as wound dressing materials because of their intrinsic properties against fungi and bacteria [59]. Ignatova et al. [45] showed that PLA nanofiber matrices alone were unable to inhibit bacteria growth (S. aureus and E. coli), but with the incorporation of chitosan or quaternized chitosan, strong antibacterial properties against the two types of bacteria were detected. One possible mechanism of this antibacterial activity results from ionic interactions between the positively charged chitosan and the negatively charged bacterial membrane, which leads to an increase of membrane permeability, subsequent cell leakage and death [45]. Besides, the chemical structure of chitosan has a high similarity to glycosaminoglycan in the ECM possessing hemostatic activity [45]. However, chitosan is not easily electrospinnable as a single polymer solution because of the chain entanglements leading to high viscosity of chitosan solutions. To account for this, other polymers like PEO or PVA are generally added to decrease solution viscosity [45, 48, 51, 56, 60-64]. Alginate is a natural occurring polysaccharide with anionic charge extracted from seaweed. Alginate dressings have hemostatic properties due to their water absorption ability (around 20 times the initial weight), and could maintain a moist microenvironment, which is especially beneficial for the early wound phases with bleeding or exudation [65, 66]. Similar to chitosan, alginate also lacks ‘spinnability’ associated with its insufficient chain entanglement at lower concentrations as well as excessive hydrogen bonding at higher concentrations. Other polymers, like PEO [67], or PVA [68] can be added to support fiber formation. Collagen is one of the native ECM components existing in skin, bone, tendon and other connective tissues. Collagen exists in a fibrillar structure with diameters in the range of 50 - 500 nm and constitutes a three-dimensional network in the native ECM. It is already well known that this specific structure contributes to cell attachment, population and proliferation. The fabricated collagen fibers normally need subsequent post-modifications in order to increase their structural stability in aqueous solutions [69, 70]. And special consideration needs to be taken in terms of denaturalization of collagen conformation as a results of high voltage during electrospinning or post cross-linking [69]. Generally, in order to obtain nanofiber matrices with a rigid structure, collagen is co-electrospun with other synthetic polymers, for example PCL [42]. Gelatin is a derivative of collagen, and widely used as food additives. Duan et al. [43] fabricated gelatin/PCL fibers in order to increase biocompatibility and mechanical properties of such dressings. The fiber mats were proved to be effective in healing skin defects. Dubsky et al. [39] showed that a

faster wound closure was obtained upon the treatment with gelatin fiber mats after 5 and 10 days administration in rat models, while no healing was observed with PCL counterparts. Other natural polymers like cellulose [71] [72] [73], hyaluronic acid [74, 75], gum tragacanth [76], zwitterionic poly (sulfobetaine methacrylate) or PSBMA [77], silk fibroin [78], zein [79], keratin [80] and soy protein isolate [81-83] also gain a lot of interest in developing wound dressings. Advantages and limitations of synthetic and natural polymers With respect to materials for wound applications, natural polymers have gained considerable interest due to their native biocompatibility, providing similar steric configuration and preceding biological simulation like molecular signal transportation and ECM-cell interaction [62]. And with the development of modern techniques, large-scale isolation of natural products with high purity from plants and animal organs is possible. However, the potential risk of immune reactions still limits their applications. Besides, in case of wound dressings, natural products often exhibit limited mechanical strength and instable structure upon contact with aqueous solutions. This contributes to the development of synthetic polymers with feasible large-scale factory production of high reproducibility. Synthetic polymers normally provide suitable mechanical properties like elasticity and stiffness, and their tunable degradation characteristics are preferred as control release carrier materials. However, compared to natural products, their biocompatibility is lower including the effect of residual solvents. Although a number of studies summarized above have attempted to combine synthetic polymers and natural products for the sake of compositing both of their advantages, new materials are still in urgent demanding in order to design systems with higher therapeutic performances and lower side effects.

5. Interactions of fibers with the wound and its environment Electrospun fibers can interact with the wounded tissue and its biological environment in multiple different ways. Fibers can affect the wound based on their chemical (polymer characteristics) and physical (fiber diameter, strength, porosity etc.) properties as well as by additional molecules attached to or incorporated into the fibers. Due to the complexity and a multitude of different effects of fibers on biological tissue, there are numerous studies published focusing on different aspects of such interactions. As these interactions can intentionally be used to facilitate wound healing and to fight negative incidences such as inflammation, infection etc. of the wound bed, the review aims to give a systematic overview about relevant studies for developing fibers for wound applications. 5.1 Interaction with cells and tissues Skin cells show specific reactions upon contacting with electrospun fibers due to the structural similarity of the fibers to the native ECM. The physicochemical characteristics of the polymer forming the fibers play an important role determining and modulating such interactions. For instance, while hydrophilic fibers generally provide an ideal surface for efficient initial cell attachment [84, 85], cell attachment onto hydrophobic fibers (e.g. based on PCL) is less efficient. A study by Planz et al. [84] demonstrated that fibroblasts proliferated significantly faster on hydrophilic PCL/gelatin fibers (water contact angle 0°) than on pure PCL fibers (water contact angle 119°) (Fig. 5, A and B). Fig. 5 Human fibroblasts seeded on electrospun fiber matrices with high hydrophobic or hydrophilic surface

Further, Fu et al. [42] studied the influence of topographical surface characteristics of nanofiber matrices on the migration activity of keratinocytes. For this purpose, two kinds of nanofiber matrices were fabricated: PCL/collagen blends with a fiber diameter of 331 ± 112 nm, featured by a rough surface and large pore size (2 µm); and the same blend matrices with an additional coating by collagen gel, resulting in an ultrafine fiber network with fiber diameters of 55 ± 26 nm, featured by smaller pore sizes (200 nm) and a smoother surface. Accelerated keratinocyte proliferation and migration was detected on collagen coated matrices, while their non-coated counterparts were not able to stimulate cell migration. The authors suggested that the smooth surface could provide more contact points (showed by vinculin staining) for keratinocytes to adhere and the small pore sizes facilitating initial cell attachment and promoting cell migration (Fig. 6). Besides, collagen coated fiber matrices exhibited the ability to activate matrix metalloproteinase-2 (MMP-2) and MMP-9, upregulate the expression of integrin ß1, Rac1 and Cdc42, and promote the deposition of laminin-332, thus accelerating the re-epithelialization process. Fig. 6 Morphology of keratinocytes on PCL/collagen fibers or collagen coated fibers

However, an inverse conclusion was drawn by Pelipenko et al. [50], based on the assumption that the mobility of keratinocytes was inhibited due to the small interfiber pore size (2560 ± 1260 nm) of PVA nanofiber matrices. They visualized that flexible cell parts (cell cytoskeleton and cytoplasm) were entrapped in the interfiber networks, while cell nuclei stayed on the surface, thus restricting cell migration on the surface as well as penetration into the three-dimensional matrices. Another study reported that the proliferation of HT-1080 cells on chitosan nano-sized fibrous matrices was lower compared to their PDLLA micro-sized counterparts caused by the smaller interfiber pore size packed densely by nano-sized fibers. As the infiltration into the fiber matrices was inhibited, the cells populated on the surface of the fibers, which can eventually be beneficial for removable superficial wound dressings [42, 62]. Besides the pore size of such fiber matrices, attention was paid to the orientation of the fibers. One study suggested that randomly oriented fibers potentially hindered cell migration [50]. Recently, a number of research groups focused on producing nanofiber matrices with aligned fiber orientation, based on the results that fibers could guide cell migration and elongation. Kurpinski et al. [46] showed that matrices consisting of aligned fibers significantly enhanced endothelial cell infiltration into three-dimensional matrices both in vitro and in vivo compared to their randomly oriented counterparts. In vivo staining of CD31+ endothelial cells and CD68+ macrophages

illustrated that both cell types infiltrated into aligned matrices to a larger extent than their randomly oriented counterparts. Chen et al. [62] fabricated dual-layer fibrous matrices combining PDLLA microfibers coated by aligned chitosan nanofibers in order to combine both advantageous features, the large pores of the microfibers and the direction of cell migration and increased specific interface area by aligned chitosan nanofibers. Improved proliferation and deeper infiltration into the threedimensional scaffolds were shown compared to the control groups, both in vitro and in vivo. Xie et al. [38] also demonstrated a faster cellular migration from peripheral towards central parts with a high expression of type I collagen on the aligned fibers compared to the randomly oriented fibers. This could be of benefit to reduce scar formation, which is caused by disorganization of newly formed ECM (mainly type I collagen fiber network). A previous study also claimed similar results showing the improvement of cell infiltration into aligned nanofibers [37]. Besides, research also illustrated that aligned nanofibers could minimize foreign body reaction frequently existing on the surface of implantation materials, which is a vital issue leading to implantable device failure [37]. Overall, the published studies demonstrate that besides the polymer properties, characteristics such as fiber morphology and orientation as well as porosity of the whole matrix can significantly affect cellular behaviors and in-depth understanding of such effects is vital for rational development of electrospun fibers for wound applications. In addition to morphological and structural effects, chemical modifications of nanofiber matrices may also alter cellular behavior. As mentioned above, additional functional molecules can be attached to the fibers to modulate their interactions with cells or for delivery of actives. For instance, Rho et al. [69] coated collagen nanofiber matrices with type I collagen, laminin and fibronectin, and studied their effects on keratinocyte adhesion and spreading in comparison with their uncoated counterparts. Such native ECM proteins contain an arginine-glycine-aspartic acid sequence (RGD), which is recognized by integrins - a protein family expressed in many cell types acting as bridges for cell - cell and cell - ECM interactions. A significantly promoted cell adhesion with a spreading cellular morphology was found for fiber matrices coated with type I collagen and laminin, while uncoated collagen matrices did not show any effect on cell adhesion. However, the adhesion capacity of matrices coated with fibronectin was barely improved. Reports by Gautam et al. [85] also showed similar results, as PCL/gelatin fiber matrices with surface grafting by type I collagen could enhance mouse fibroblasts (L929) adhesion and proliferation. However, results from Chutipakdeevong et al. [78] demonstrated that surface immobilized with fibronectin on silk fibroin nanofiber matrices could obviously improve human fibroblasts adhesion, viability and three dimensional migration in vitro. Kurpinski et al. [46] showed heparin immobilized nanofiber matrices improved cell infiltration based on both, random and aligned structures. In vivo staining of CD31+ endothelial cells and CD68+ macrophages illustrated that both cell types infiltrated into heparin conjugated matrices to a higher extent than into their uncoated counterparts. One possible reason resulted from the anti-clotting property of heparin, which may allow more cells migrating throughout the matrices. Moreover, the property of heparin binding to several ECM proteins (fibronectin) and growth factors could facilitate cell adhesion, proliferation and migration. The interplay of electrospun fibers and cells / tissues is extremely complex and depending on the polymer characteristics and the fiber morphology and the properties of individual fibers as well as of the three-dimensional fiber matrix. In addition, comparability of published studies is often impaired as every research group uses individual test method and equipment. However, based on an in-depth understanding of fiber - cell interactions for a specific system, tailored wound dressings can be fabricated and offer tremendous opportunities for future therapy. 5.2 Interaction with wound bacteria As the physical barrier of intact skin is impaired in case of a wound, pathogen invasion mostly by

bacteria frequently occurs. Besides the risk of developing a generalized infection encroaching on other organs, invading bacteria can turn an acute wound into a chronic wound with impaired healing. In fact, 60% of infections are found in chronic wounds, comparing to only 6% in acute wounds [86, 87]. Electrospun fibers can also interact with bacteria and there are plenty studies investigating such effects in vitro. These studies often stem from research areas like material sciences and aim to investigate colonization of fibers for filters and biomedical applications like implants. Bacterial colonization and biofilm formation normally start from bacterial attachment to interface or substratum of nanofiber matrices. Said et al. [17] found a large number of bacterial cells on the surface of PLGA nanofiber matrices and a dense biofilm inside the pores after 24 hours’ incubation in a broth culture containing wound isolate methicillin-resistant Staphylococcus (MRSA1). The population of biofilm bacteria reached about 4.375x107 CFU/cm2 of the initial concentration of 108 CFU/ml. Bacterial growth can be prevented by applying polymers with antimicrobial properties like chitosan. For instance, Ignatova et al. [45] demonstrated attachment of S. aureus to the surface of PLA nanofibers after incubation for 24 hours, while no bacteria was found on cross-linked chitosan/PLA fibers. 6. Delivery of Active Pharmaceutical Ingredients (APIs) As introduced in chapter 3, various strategies could be applied to incorporate drugs with different physicochemical properties into electrospun fibers. The following paragraphs give an overview about drug-loaded electrospun fibers for wound application loaded with different classes of actives. 6.1 Antiseptics Among a plentitude of different antiseptics for wound care, mostly alcohols, benzalkonium chloride, hydrogen peroxide and iodine, there have already been attempts to load silver into electrospun fibers. Silver and silver ions have a broad-spectrum biocidal effect against almost 650 strains of bacteria and thus have extensively been used in clinical treatments for burns and chronic wounds for centuries [88]. The exact mechanism of action against microorganisms is not completely understood yet, changes in bacterial cell morphology and structure have been hypothesized. Silver can penetrate bacterial cell membranes and attack their respiratory chains, thus causing bacterial death. Silver ions may change the state of DNA to its condensed form and deactivate its replication ability leading to cell death [8992]. By increasing the surface via silver nanoparticle generation, the increased surface to volume ratio results in a larger contact area to the microorganisms and thus promoting antibacterial properties. Besides, silver nanoparticles (NPs) were found to release silver ions, which also contribute to the increasing antibacterial activity [89]. Silver NPs have been loaded into nanofiber matrices through a number of methods. Xu et al. [93] prepared nanofiber matrices by electrospinning a single solution consisting of AgNO3 and poly(ethylene-co-vinyl alcohol) (EVOH). Then, silver NPs were obtained by deoxidization through post-exposing of nanofiber matrices to illumination and UV lights. A linearly increasing antibacterial effect associated with silver loading concentrations was found, that the highest silver concentration tested produced the largest bacteriostatic loop, indicating the strongest bacterial killing capacity. Rivero et al. [34] obtained silver NPs loaded fiber mats by immersing poly(acrylic acid) nanofibers in 0.1M AgNO 3 solution for 5 min, Ag+ was loaded with available carboxylic groups of poly(acrylic acid) by ion exchange, then Ag+ was reduced to Ag NPs by 0.1 M dimethylamine borane solution. Another method by Lakshman et al. [94] comprised synthesis of silver NPs followed by subsequent electrospinning of a PU solution with suspended silver NPs. Jin et al. [95] investigated the influence of the amount of silver NPs on cell proliferation and antibacterial activity. They fabricated PLLCL nanofiber matrices loading silver NPs with various concentrations ranging from 0.25% to 0.75% (wt%). Similar to the results published by Xu et al. [93], their antibacterial activity against S. enterica and S. aureus increased with the increasing amount of loaded silver NPs. However,

human skin fibroblasts showed better proliferation and comparable morphology to those on tissue culture plates after co-culture with matrices containing less silver NPs (0.25%, wt%). Li et al. [96] proved that Ag NPs coupled with chitosan oligosaccharides loaded PVA nanofiber matrices could accelerate wound healing in early stages in vivo without skin irritation. Besides, silver NPs could also be delivered along with other APIs like olive oil as wound dressings [97], and silver ions were also incorporated as salts, such as silver acetate [98], or silver-sulfadiazine [99, 100]. 6.2 Small molecules Small molecular drugs with various biological / pharmacological activities have been incorporated into electrospun fiber systems mostly with the intention to combat wound inflammation and infection. Such molecules include antibiotics, anti-oxidants and anti-inflammatory agents, endogenous signaling molecules, herbal extracts etc. The table below summarizes these studies, while some examples are discussed in more detail in the following paragraphs. Jannesari et al. [16] spun a single solution containing PVA, poly(vinyl acetate) (PVAc) and ciprofloxacin HCl, and investigated parameters affecting the drug release. By blending the hydrophilic polymer PVA with the hydrophobic polymer PVAc, the weight loss of the fiber mats was reduced and their physical stability was improved. The drug was released in a controlled way for 250 hours. The study also showed that the thickness of the fiber mats correlated with the release kinetics, as for thick fiber mats the release was slower with decreased initial burst effect, which was explained with a lower degree of swelling leading to a slower drug diffusion out of fiber mats. Kataria et al. [101] studied the in vivo healing properties of ciprofloxacin loaded PVA/sodium alginate fiber mats in rabbits, and a faster wound area reduction was found with the treatment of ciprofloxacin loaded formulation compared to the non-loaded group. However, the difference in wound healing between the two groups was not significant, which indicated ciprofloxacin as an antibiotic, could de-contaminate wound bed, but has little effect to promote acute wound healing. Said et al. [17] found that with 10% of fusidic acid loading, PLGA nanofiber matrices could eradicate planktonic bacteria (S. aureus ATCC 6538P, Ps. Aeruginosa ATCC 9027, and a methicillin-resistant S. aureus clinical isolate (MRSA1)) and also significantly suppress MRSA1 biofilm formation by 90%. With incorporation of the antibiotic fusidic acid into PLGA nanofiber matrices, biofilm formation was significantly suppressed (decreased to around 10% of the maximal bacterial population of biofilm). And incubation with bacteria in reverse increased the initial burst release of fusidic acid, which was presumed to be the alteration of surface properties or structural integrity of PLGA nanofiber matrices as a result of bacterial colonization. One possible mechanism may be the hydrolysis of PLGA catalyzed by lipolytic esterases secreted by both bacterial species (Ps. aeruginosa and S. aureus). Alhusein et al. [102] evaluated the capability of tetracycline HCl loaded triple-layer fiber matrices (consisting of poly(ethylene-co-vinyl acetate) and PCL) in disposition of biofilm. The fiber matrices showed efficient activity in preventing biofilm formation and killing formed biofilm by S. aureus MRSA252 attributed by the sustained release of antibiotics. A similar (at day 4) and significantly larger inhibition zone (at day 5) were exhibited compared to commercial disc. Furthermore, a colony biofilm model which simulated biofilm growing in real wound environment was applied, and results showed that the matrices could decrease the viable count of bacteria by 80% in a 72 h mature biofilm. For treating inflammation of the wounded area, Cantón et al. [103] prepared ibuprofen loaded PLGA nanofibers, which could evidently reduce the inflammatory activities of fibroblasts by reducing LPS stimulated NF-kB translocation. Curcumin has already been investigated for its efficiency against impaired wound healing based on its wide range of biological properties including anti-inflammatory, anti-oxidant and anti-bacterial activity. Panchatcharam et al. [104] assumed the promotion of wound healing of curcumin resulted from its anti-oxidant activity, based on the results of increasing level of glutathione peroxidase, superoxide dismutase and catalase while decreasing level of lipid peroxides with curcumin treatment, leading to a faster maturation of collagen. Fu et al. [18] prepared

curcumin/PCL-PEG-PCL electrospun fiber mats with a sustained release for 10 days, and an increasing deposition of collagen, a faster new blood vessel formation and re-epithelialization rate were achieved with the treatment of such fibers. Ramalingam et al. [105] also showed the antibacterial property of curcumin loaded poly(2-hydroxy ethyl methacrylate) p(HEMA) nanofiber against MRSA and extended spectrum ß lactamase. Based on the long history of treating skin wounds with herbal extracts, researchers tried to encapsulate them into electrospun fibers with the aim of long-term release. Jin et al. [19] successfully formulated nanofibers based on PCL and extracts from four traditional Indian medical plants, Azadirachta indica, Indigofera aspalathoides, Myristica andamanica and Memecylon edule (ME). Through the incorporation of the individual plant extracts, the wettability of fiber mats significantly increased with water contact angle in the range of 17o - 23o, while PCL fiber mats showed high hydrophobicity (136o). Fibroblasts showed a faster proliferation on PCL/ME fiber mats with a spindle shape compared to the other fibers loaded with the extracts as well as pure PCL fibers. And a larger amount of collagen secreted from PCL/ME treated fibroblasts was obtained. Besides, Jin et al. also tested the ability of PCL/ME as a stem cell niche to direct adipose tissue-derived stem cells epidermal differentiation. Ker 10 and filaggrin as two chosen epidermal markers were observed throughout the whole cells with polygonal and round morphologies after their 15 days’ culture on PCL/ME nanofibers. The results proved PCL/ME to be a strong candidate for wound healing and directing of stem cells differentiation. Motealleh et al. [106] electrospun PCL/polystyrene fibers including extracts from chamomile plants, which contained mainly apigenin. The matrices with 15% of chamomile loading showed antimicrobial properties against S. aureus and C. albicans with inhibitory zones around 7.6 mm. After 14 days post-wound treatment, chamomile loaded matrices showed a higher extent of wound closure compared to commercial products, 11% higher than the commercial product Com®feel (without silver particles) and 19% higher than a gauze bandage. Nitric oxide (NO) is generated in the human body and plays a crucial role in a number of physiological functions. One of factors leading to diabetic chronic wounds is on account of insufficient NO production. NO can modify early response genes and thus influences wound healing. Besides, NO shows antimicrobial activity as well. Despite its advantages, the clinical application of NO is limited due to its gas state. Lowe et al. [107] succeed in binding NO to the backbone of acrylonitrile-based terpolymers nanofibers. NO bonding was proved by infrared spectroscopy and its release profile was further quantified by the Griess assay. The dressings could release 79 µmol NO g-1 over 14 days. Cutaneous implantation of NO loaded dressings for 30 hours increased the expression of the early response genes, namely cFos, JunB and eNOS, while only JunB expression showed a statistical significant increase. However, treatment with NO loaded dressings did not change the level of vascular endothelial growth factor (VEGF) expression. The accelerated wound healing was proved after 1 week by applying the NO loaded dressings to a rodent model in a weekly manner, and daily application with these dressings could promote healing process faster, with statistical significance at day 4. The possible mechanism for accelerating wound healing was NO-induced angiogenesis, proved by a 60% higher capillary density compared to the control groups. As mentioned in chapter 1, diseases like diabetes are one of factors leading to delayed wound healing. It has already been found that the resistance to insulin could delay the process of re-epithelialization and contraction, thus impairing cutaneous wound healing. With the fact that metformin could enhance biological activities of insulin and its receptor, application of metformin has been considered as another therapeutic strategy for diabetic ulcers. Lee et al. [108] used PLGA as carrier material to load metformin. The incorporation of metformin increased the hydrophilicity of the fiber matrices from 104.83o to 47.47 o, with an increasing water uptake capacity from around 94% to 160%. The release of metformin in vitro and in vivo could last for three weeks. And a significantly faster wound reepithelialization and closure was achieved by metformin loaded matrices compared to non-metformin loaded fiber mats or gauze. Lee et al. [109] further loaded metformin hydrochloride into

collagen/PLGA matrices, which showed a relatively similar wound accelerating behavior. Furthermore, collagen expression levels were compared among three treatments, that type I collagen exhibited a significantly higher content in dermis by the treatment of loaded matrices (1.06 ± 0.03) than blank matrices (0.36 ± 0.02) or gauze (0.13 ± 0.02). This was found to be contributed by the inhibition of matrix metalloproteinases (MMPs), which are major enzymes for extracellular degradation. As metformin hydrochloride could significantly inhibit MMP-9 expression, collagen was protected from degradation. These studies provided a new solution for the therapy of chronic wound concerned with diabetes. Other small molecules with multiple pharmacological properties were also combined with electrospun fiber systems for wound healing, like genistein and epigallocatechin-3-O-gallate with antiinflammatory, antioxidant, antibacterial activities or immunomodulatory activities [110, 111], and 20(R)-ginsenoside Rg3 with the ability of prevention of hypertrophic scars by interfering with angiogenesis procession [112], which all showed positive results for further studying. Table. Electrospun fibers loaded with small molecular drugs for applications to skin wound

6.3 Macromolecules Macromolecules are always challenging to be delivered to the pharmacological target due to their fragile structure and sensitivity to organic solvents. Thus, the fabrication of electrospun fibers loaded with pharmacologically active macromolecules requires sophisticated drug incorporation techniques such as emulsion spinning and co-axial spinning. For the purpose of facilitating wound healing, lysozyme, growth factors, growth factor-encoding plasmids and even cells have been incorporated into electrospun fibers. Lysozyme Lysozyme is a water soluble protein consisting of 129 amino acid residues, a member of glycoside hydrolases with roles including degradation of polysaccharide. The antimicrobial action of lysozyme is based on hydrolysis of the linkages between peptidoglycans and chitodextrins in the cell wall of bacteria and fungi [113]. Charernsriwilaiwat et al. [114] delivered lysozyme to skin wound through chitosan/PVA based nanofiber matrices. The design was based on the antibacterial activity of lysozyme and the degradation byproducts from chitosan, namely N-acetyl-D-glucosamine, which have been proved to initiate the proliferation of fibroblasts and accelerate wound healing [115]. In vivo wound healing studies were performed in a rat model, by comparison of lysozyme loaded chitosan/PVA fiber mats with gauze on the one hand and commercial antibacterial gauze on the other hand. Lysozyme loaded fiber mats showed a better healing effect than gauze, and a similar effect as antibacterial gauze. However, more profound studies should be carried out to prove the interaction of lysozyme and chitosan playing a role in assisting wound healing. Growth factors As already mentioned in chapter 1, growth factors play a crucial role in regulating wound healing processes. Upon wounding, large amounts of growth factors are released from keratinocytes, fibroblasts, platelets and macrophages and subsequently direct cell behaviors. Behm et al. [116] reviewed the function of mediators including growth factors in wound healing. In early wound phases, keratinocyte growth factors (KGF) are produced by fibroblasts, protecting keratinocytes against ROSinduced damage through enhancing the expression of intracellular enzyme peroxiredoxin-6. KGF also targets NF-E2-related factor 2 (Nrf2), generating detoxifying enzymes and antioxidant substances in keratinocytes. Transforming growth factor β1 (TGF-βI) binds to TGF-β receptor II resulting into the recruitment of macrophages and inflammatory cells to remove cell debris. Besides, TGF-βI induces

fibroblast proliferation and migration, increases collagen production and contributes to angiogenesis and the formation of granulation tissue. Further, vascular endothelial growth factor (VEGF) promotes angiogenesis. Epidermal growth factor (EGF) family members such as EGF, transforming growth factor-alpha (TGF-α) and heparin binding EGF, initiate keratinocyte proliferation and migration through binding to their EGF receptors expressed on keratinocytes. Fibroblast growth factor (FGF) contributes to re-epithelialization, stimulation of fibroblast proliferation and migration. In addition, growth factors are also involved in the regulation of MMPs, which are important enzymes for ECM remodeling and angiogenesis [116]. Based on the crucial parts growth factors play in various wound healing stages, many groups incorporated growth factors as actives into wound dressings. Bertoncelj et al. [20] investigated the influence of platelet-rich plasma (PRP) on cell proliferation by supplementing PRP with various concentrations from 0.05% - 15% (v/v) to cell medium. Results showed that a small amount of PRP like 0.05% was enough to stimulate metabolic activity of keratinocytes, while concentrations higher than 5% inhibited their metabolic activity. Fibroblasts exhibited a concentration-dependent metabolic activity associated with concentration ranging from 0.5% - 2%, with the PRP amount outside of this range exerting a negative effect. Then PRP was incorporated into nanofiber matrices consisting of chitosan and PEO though electrospinning a single solution. Nanofibers released around 70% of the proteins during the first three hours and complete release within 24 hours. Both, keratinocytes and fibroblasts, showed an increasing metabolic activity when cultured with PRP loaded matrices compared to blank matrices. And a synergistic promoting effect for cell proliferation was obtained from nanotopography and PRP. However, cells co-cultured with blank nanofibers in a medium supplemented with 2% of PRP showed the most significant response compared to PRP incorporated matrices, since the limited amount and fast release of loading PRP from nanofibers. These results shed light on further studies focusing on increasing PRP loading and release in a controlling profile. Gümüşderelioğlu et al. [28] found a significantly higher expression levels of loricrin in human dermal keratinocytes after cultivation with EGF immobilized matrices, indicating its ability for the maintenance of keratinocytic phenotypes. The results were consistent with a previous study [31]. Yang et al. [24] embedded basic FGF (bFGF) into ultrafine nanofibers. They exerted a core-shell structure with a low burst release (14%) and gradual release for four weeks. Contributed by the controlled release profile, the viability of mouse embryo fibroblasts was remarkable higher than that of blank matrices with or without supplement of bFGF (control groups). And secretion of collagen Type I from bFGF loaded matrices was considerably higher. In vivo studies were performed based on diabetic rat model. A faster wound healing progress was obtained with the treatment of bFGF loaded matrices than in control groups, with faster local scab and granulation tissue formation and a faster skin regeneration with hair growth, no infection was existing. While in control groups, remarkable inflammation was observed with a delayed wound healing. In addition, regenerated skin treated by bFGF loaded matrices possessed a similar structure with normal skin, which suggested bFGF loaded matrices exerting the anti-scar potential. As growth factors are engaged in certain functions during different healing stages, another strategy for the delivery of multiple growth factors in a programmed manner in order to provide them for a certain healing phase has been developed with a nanoparticle-in-nanofiber system. In general, growth factors needed for the early stage of wound healing were encapsulated into the nanofiber matrices composed of fast degradable polymers, while growth factors for late stage of wound healing were incorporated into nanoparticles first, followed by further incorporation into nanofiber matrices. The aim was a rapid release of growth factors for the early promotion of epithelialization and angiogenesis followed by tissue regeneration and remodeling to be regulated by the later release of growth factors. Xie et al. [117] fabricated this kind of system by encapsulating VEGF into PLGA nanoparticles, then the nanoparticles were embedded into platelet-derived growth factor-BB containing chitosan and PEO nanofiber matrices. Lai et al. [118] designed a similar system which aimed for the delivery EGF and

bFGF for the promotion of early stage of epithelialization and angiogenesis, then PDGF and VEGF were delayed released expected to assist blood vessel maturation. Both of them obtained promising results from in vitro cell study and in vivo rat wound model. Plasmids In addition to growth factors, therapeutic application of genes encoding growth factors poses an alternative strategy. Yang et al. [25] firstly generated polyplexes of bFGF-encoding plasmid (pbFGF) with poly(ethylene imine) (PEI) via electrostatic interaction. The polyplexes were further emulsified with poly(DL-lactide)-poly(ethylene glycol) (PELA) solution and electrospun into nanofiber matrices. pbFGF loaded nanofiber matrices showed a core shell structure with pbFGF in the core and high encapsulation efficiencies in the range of 79.3% - 87.7% were obtained. 10% of PEG with molecular weight of 2 or 4 kDa was included in order to modulate the sustained release of pbFGF for 4 weeks. Mouse embryo fibroblasts showed a significantly lower attachment to pbFGF loaded nanofiber matrices compared to tissue culture plates (TCP), and the released pbFGF polyplexes developed remarkable cytotoxicity. While pbFGF polyplexes exerted inhibition of cell growth, the continuous release and expression of bFGF succeeded in promoting cell proliferation, which led to the cell growth rates on pbFGF loaded nanofiber matrices similar to that on TCP. In vivo tests with diabetic rats showed a nearly completed wound recovery within 3 weeks with pbFGF polyplexes loaded fiber matrices, while the open wound area after treatments with nanofiber matrices supplemented with polyplexes, blank nanofiber matrices and no treatment resulted in 13%, 22% and 33% of initial wound bed size, respectively. Wound treated with pbFGF polyplexes loaded fiber matrices also showed an improved collagen production and tissue regeneration, contributed by the gradual release and expression of pbFGF. Kim et al. [119] made use of MMPs, existing at impaired wound sites with abnormally elevated levels, as the trigger to release plasmid human epidermal growth factor (phEGF) from carrier systems. phEGF was immobilized onto PEI conjugated on the surface of nanofiber with an MMP-cleavable peptides. In the presence of MMPs, phEGF and PEI complexes were released through the digestion of PEI and nanofiber linker, with more efficient cell transfection compared to naked phEGF. High level of hEGF expression was quantified at day 14 in wound site without contraction by the treatment of phEGF loaded matrices. Further, wound recovery was accelerated without phenotypic changes of proliferative keratinocytes, proved by co-expression of keratin 5 and keratin 14. Besides the most widely used macromolecules, Zhang et al. [120] delivered monocyte chemoattractant protein-1 with heparinized PCL and chitosan matrices to successfully enhance local angiogenesis. And Macri et al. [21] incorporated FNIII1-derived P12 into tyrosine-derived polycarbonate terpolymer nanofiber matrices as potential wound dressings. 6.4 Cells Besides the delivery of pharmacologically active substances, cells can also be applied for therapeutic purposes. This holds especially true for large burn wounds, for which the clinical gold standard is still autologous skin transplantation. However, limitations for harvesting sufficient numbers of cells from the patient´s body evoked a clinical need for tissue-engineered skin substitutes as an alternative. To produce such substitutes, skin biopsies must be taken from patients, followed by isolation of the skin cells including keratinocytes and fibroblasts. Those cells are subsequently seeded on suitable scaffolds after proper in vitro expansion. For full-thickness skin substitutes, keratinocytes must be seeded after the dermal parts have been established by fibroblasts as the supporting matrix. Such skin substitutes are already commercially available for clinical use subdivided into epidermal, dermal and epidermal/dermal substitutes which have been reviewed by previous publications [10, 121, 122]. Although tissue-engineered allogeneic skin substitutes bear the risks of transmitting viruses and inducing rejection by the immune system, the relatively low costs and potential abundant availability

contribute to their promising development [122]. For the potential application of electrospun nanofibers in tissue-engineered allogeneic skin substitutes, several studies attempted to cultivate stem cells guided to differentiate into skin cells like keratinocytes or fibroblasts for wound healing. Stem cells therapy is promising in terms of providing complex signaling molecules facilitating natural wound healing process. Scaffolds composed of PLLA/collagen, PLCL/polyxamer or PVA/gelatin etc. have been investigated for stem cell cultivation [123-127]. For the guidance of stem cell differentiation into certain cell types, special inducers are demanding. For example, in order to achieve epidermal differentiation, inducers like EGF, insulin, 3,3’,5-triiodo-L-thyronine, hydrocortisone and 1α, 25dihydroxyvitamin are supplemented into medium [128], which possess challenges for stem cell therapy since in vivo environment is complicated and uncontrollable to certain extent. Besides inducers, a number of additional parameters have an influence on stem cell behaviors, such as matrix elasticity [129]. 7. Conclusions and Outlook Electrospun fiber mats have gained considerable interest as functional wound dressings providing mechanical protection while at the same time preventing invasion of bacteria, allowing for gas exchange as well as a moist wound environment. The fabrication of electrospun fibers is feasible with a number of commercially available polymers providing a wide range of physicochemical properties. Further, the fibers can be functionalized by attaching molecules to their surface to improve interactions with skin cells and tissue. By applying sophisticated incorporation techniques, loading and controlled release of actives is possible, even for fragile macromolecules. Notably, electrospun fibers do not only serve as inert carriers for drug delivery, but also actively interact with the wound environment and can thus even facilitate wound healing. However, even though electrospinning is a versatile platform technology, the development of an optimized and reproducible drug-loaded fiber formulation is challenging. More in-depth understanding and standardization of this technique are required, especially for advanced spinning approaches as emulsion and coaxial electrospinning. Further, the upscaling of a distinct formulation is a mandatory prerequisite for industrial production. Thus, the development of complex therapeutic fibers entails the development of sophisticated big-scale production devices. For rapid translation of such systems to the patients in clinics, suitable and predictive test methods have to be established and validated to allow for reproducible and comparable evaluation of such fiber mats among different research facilities and subsequently production sites. In conclusion, electrospun fiber mats have already proved themselves as innovative wound dressing candidates with a high therapeutic potential. With continuous development of this technology, more studies can be expected with improvement in performance of electrospun fibers, and deeper understanding of their role in wound healing processes.

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Acknowledgments Jing Wang thanks the China Scholarship Council for her PhD fellowship.

Conflict of Interests Statement The authors declare no conflicts of interest.

Correspondence Prof. Dr. Maike Windbergs Institute of Pharmaceutical Technology and Buchmann Institute for Molecular Life Sciences Goethe University Frankfurt

Max-von-Laue-Str. 15 60438 Frankfurt am Main, Germany Phone: [0049]-(0)69-798-42715 Email: [email protected]

Table and Figure Legends Table. The summary of small molecular drugs for skin wound healing. Fig. 1. Schematic illustration of four wound healing phases. (A) clot formation to stop bleeding, (B) endogenous substances defending and signaling molecules transportation, (C) re-epithelialization and granulation tissue formation, (D) ECM remodelling. Fig. 2. Morphology of electrospun fibers (A) and ECM (B) based on scanning electron microscopy (SEM). (Adapted from Planz et al. [84] by permission of The Royal Society of Chemistry). Fig. 3. Systemic setup for electrospinning (A), a presentive photograph of fiber mats (B) and surface morphology under SEM (C). Fig. 4. Schematic illustration of drug incorporation strategies. (A) blend electrospinning, where drugs and polymers are co-dissolved in solvents to be spun; (B) emulsion electrospinning, where drug solutions are emulsified into immiscible polymer solutions, followed by spinning; (C) co-axial

electrospinning, where drug and polymer solutions are separately spun through two concentric nozzles; (D) post-immobilization, where drugs are conjugated onto fabricated nanofiber matrices through physical or chemical interaction. Fig. 5. Human fibroblasts seeded on electrospun fiber matrices with high hydrophobic surface (A), and hydrophilic surface (B) (Adapted from Planz et al. [84] by permission of The Royal Society of Chemistry). Fig. 6. The morphology of keratinocytes seeded on PCL/collagen (A, C, E and G) and PCL/collagen with collagen gel coating (B, D, F and H) (Immunofluorescent staining of F-actin (Red), nuclei with DAPI (purple/blue) and vinculin (green)), I) Percentage of polarized keratinocytes, n=3 (Reprinted from [42] with permission from Elsevier).

Table. The summary of small molecular drugs for skin wound healing Target Type

Drug ciprofloxacin

antibiotics

fusidic acid polyhexamethylene biguanide berberine cetyltrimethylammonium bromide tetracycline HCl nisin neomycin ampicilin amoxicillin

Carrier polymers PVA/poly(vinyl acetate) PVA/alginate PLGA Cellulose acetate and polyester urethane Collagen and zein

Ref. [16] [101] [17]

polyvinylpyrrolidone

[131]

PCL/PEVA PEO/PLA PSSA-MA/PVA PU PLGA

[132] [133] [134] [135] [136]

[130] [79]

clarithromycin curcumin anti-oxidant and antiinflammation

ibuprofen dexamethasone sodium phosphate piroxicam

NO

nitric oxide

iron chelator

2,3-dihydroxybenzoic acid

anti-diabetic

metformin emodin shikonin

herbal extracts

Others

Indigofera aspalathoides, Azadirachta indica, Memecylon edule, Myristica andamanica tea tree oil phaeodactylum tricornutum Tecomella undulata chamomile thymol herbal centella asiatica sedum dendroideum genistein astragaloside IV epigallocatechin-3-Ogallate (EGCG) ginsenoside Rg3

PLA PCL-PEG-PCL Poly(2-hydroxy ethylmethacry-late) PLGA

[137] [18] [105] [103]

PVA/honey

[138]

PCL-PVAc-PEG Acrylonitrile-based terpolymers Poly(lactic-co-glycolic cohydroxymethyl propionic acid)

[139] [107]

PLA/PEO

[141]

PLGA Collagen/PLGA PVP cellulose acetate, poly(L-lactide), PLGA PCL/poly(trimethylene carbonate) (PTMC)

[108] [109] [142]

PCL

[19]

PCL gelatin

[145] [146]

PCL/PVP PCL/PVP PCL/PLA Cellulose acetate PLA PLA/PEO silk fibroin/gelatin

[147] [106] [148] [149] [150] [110] [151]

PLGA

[111]

PLGA

[112]

[140]

[143] [144]

Graphical abstract: