Wound Dressing from Electrospinning

Electrospun mesh with its high-surface area, porosity and flexibility in material selection and additives loading makes it attractive for use as wound dressing [Abrigo et al 2014]. Factors such as fluid update ability and water vapor transmission rate are some parameters that affect the performance of wound dressing through maintaining a moist environment while removing excess exudates. Wound dressings that are highly flexible are also useful for wounds on "difficult" anatomical sites. These properties are in turn influenced by material selection, structure and form of the dressing [Jones et al 2006]. The typical electrospun output comes in the form of a flat nonwoven film and its flexibility allows it to be used in most wound sites. Electrospinning may also be combined with other manufacturing processes to tailor the properties of the wound dressing. Al Kayal (2020) constructed a bilayered fibrin/poly(ether)urethane (PEU) scaffold with PEU phase-inversed membrane as the base supporting the electrospun layer of fibrin fibers. After electrospinning of fibrin, polymerization was obtained by thrombin solution spraying onto it. Adhesion of the fibrin fibers on the PEU base was found to be strong.

General Performance

The first requirement of wound dressing is to maintain a favourable environment for healing. This involves maintaining a moist environment while removing excess exudates. Wang et al (2015) tested the performance of electrospun poly(ethylene-co-vinyl alcohol) (EVOH) on its fluid update ability and water vapor transmission rate (WVTR). Using distilled water, the fluid update of electrospun EVOH was found to be more than 80% while that of cotton gauze is about 68%. This shows that electrospun EVOH has a higher potential to cope with more exudates compared with cotton gauze. In terms of water vapor transmission rate (WVTR), it has been suggested that WVTR between 2000 g.m^-2.24h^-1 and 2500 g.m^-2.24h^-1 is preferred [Queen et al 1987]. Distilled water is commonly used in these experiments and a comparative study by Queen et al (1987) showed that the difference in WVTRs from experiment conducted using water and plasma is insignificant. With electrospun poly(ethylene-co-vinyl alcohol) (EVOH), the WVTRs were from 900 to 1000 g.m^-2.24h^-1 [Wang et al 2015] which is lower than the recommended WVTR. Since the selection of material will have a significant influence on WVTR, Motealleh et al (2014) used a composite of electrospun poly(e-caprolactone)/polystyrene (PCL/PS) instead. Its WVTR was found to be 1896 g.m^-2.24h^-1 which is very close to the recommended WVTR. On the other hand, electrospun poly(e-caprolactone) loaded with 2% tetracycline hydrochloride showed WVTR in the range of 3290-3378 g.m^-2.24h^-1 [Chellamani et al 2014].

Several studies have shown that cell migration is faster on oriented fibers than cast films [Shang et al 2010, Liu et al 2010]. In wound dressing, it is advantages for quicker cell coverage on the wound to facilitate recovery. Shin et al (2019) used a modified electrospinning setup to create radially patterned polycaprolactone (PCL) nanofibers to encourage faster migration of human bone marrow stem cells from the periphery to the centre of the scaffold. Comparison of cell migration speed was made with randomly oriented electrospun PCL nanofibers. Both radially and randomly oriented nanofibers have a diameter of about 380 nm and interfiber pore size of about 12 nm. From the SEM images of the scaffolds, the radial patterning of the nanofibers are not very clear. However, there are distinct difference in the migration speed of the cells on the radially patterned and randomly oriented nanofibers scaffolds. From day 5 of the cell culture, there is a difference in the cell coverage on the scaffolds. On day 7, the randomly oriented nanofiber scaffold showed a median cell-free area of 84% and the radially patterned nanofibrous scaffold showed a median cell-free area of 49%. This clearly showed the benefits of having radially patterned nanofibers in guiding the cells towards the centre of the scaffold.

Drug Release

In wound dressing for more serious cases and inflammatory skin disorders, the ability to encapsulate drugs into the dressing will enhance healing and treatment of the injured site. Brooker et al (2021) used electrospun poly(ethylene oxide) (PEO) fibres for encapsulating antioxidant and anti-inflammatory nanoparticles composed of crosslinked poly(propylene sulfide) (PPS). The electrospinning was carried out in an aqueous medium since PEO is a water soluble polymer. This electrospun PEO/PPS-NPs membrane is targeted for dry wound disorder hence the release rate of the drug will be slow and the wound dressing will have a moisturising effect. Up to 14.3% PPS-NPs has been loaded into PEO solution and electrospun into fibers with average fiber diameters of 191 nm and 166 nm for neat fibers. The PEO/PPS-NPs was found to be biocompatible when tested on human dermal fibroblasts. The PPS-NPs from the electrospun fibers were also found to retain its anti-inflammatory properties as demonstrated by TNF-α inhibition from lipopolysaccharides stimulated RAW264.7 macrophages. Yue et al (2022) used electrospinning to encapsulate liposomes prepared with astragaloside IV (AS), and astragalus polysaccharide (APS) in polyvinyl alcohol (PVA) fibers. Both AS and APS were extracted from Astragalus membranaceus, a major herbal remedy for diabetic foot ulcers used in traditional Chinese medicine. Preparation of the solution for electrospinning is through blending of the active ingredients into the PVA solution. Efficacy of the membrane was tested in vivo on SD rats with full thickness wound. PVA membrane loaded with AS liposome (ASL) and APS was found to have less wound inflammation, greater deposition of collagen and regeneration of epithelium compared to pure PVA membrane and negative control. At day 15, the wound was completely covered in the groups with ASL/APS/PVA membrane and APS/PVA. The group with ASL/APS/PVA membrane showed the best recovery with the most closely arranged collagen fiber deposition.

Bacteria Exclusion

Typical pore size of electrospun fibrous membrane is less than 5µ and this is able to prevent bacteria from reaching the wound. To demonstrate the effectiveness of electrospun layer in filtering out microbes and bacteria, Lev et al (2012) first electrospun a layer of polyurethane over a polypropylene supporting substrate. With a electrospun nanofibers weight of 3.8 g/m², it is able to filter out E. coli although at weight less than this, E. coli was able to get pass the membrane. Chaudhary et al (2014) used an electrospun polyacrylonitrile-silver composite filter media to cover a nutrient media in room condition and passes ambient air through the filter media. When compared to the negative control which is without the protective filter media, the nutrient media protected by the nanofibrous filter remains free of bacteria growth after two months while the unprotected nutrient media show microorganism growth.

Anti-bacterial

In wound dressing application, it is essential that factors that give rise to its anti-bacterial property must not compromise wound healing. Cai et al (2010) fabricated chitosan/silk fibroin composite nanofibers using electrospinning and tested for its anti-bacterial property and biocompatibility. While the composite material showed inhibition effect on E. coli, it does not seem to be effective against S. aureus. Biocompatibility study using murine fibroblast showed that the composite membrane promotes cell adhesion and proliferation. To prevent wound infection, the use of antibiotics is the most direct method. However, frequent use of antibiotics have led to increasing antibiotic resistance. Other methods of preventing infections have been explored to reduce the reliance of antibiotics. Bao et al (2022) used a combination of graphene (Gr) and polyphenolic tannic acid (TA) in electrospun hyaluronic acid (HA) for the construction of a wound dressing. Gr is thought to exhibit antibacterial effects by physical puncturing and cutting, oxidative stress-induced damage, and photothermal effects. TA is also known to exhibit strong antibacterial properties and crosslink with Gr through hydrogen bonding or ions. Further TA can inhibit hyaluronidase activity, to enhance the stability of hyaluronic acid To create the antibacterial wound dressing, TA and Gr were added to a solution of HA and electrospun to form the membrane. The resultant membrane undergoes photocrosslinking using a solution of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and UV irradiation. When tested against Staphylococcus aureus, HA/Gr membrane has an inhibition rate of 38% while pure HA membrane has an inhibition rate of only 8%. HA/Gr/TA membrane has the highest inhibition rate of 97%. Therefore, while Gr exhibits antibacterial property, the addition of TA is necessary to bring the antibacterial effect to over 90%. Biocompatibility tests were carried out using NIH-3T3 cells on a composition of HA/Gr/TA (20% w/v HA + 0.1% w/v Gr + 0.3% w/v TA) and compared with the control which were cells cultured in the well without any membrane. Their results showed that the proliferation of the cells on the HA/Gr/TA membrane was significantly better than the control over a 36 h period which provided evidence that the Gr at such low concentration may be safe.

Commercially available drugs may be loaded into electrospun fibers to introduce anti-bacterial property while ensuring biocompatibility. Unnithan et al (2012) loaded polyurethane/dextran nanofiber mats with ciprofloxacin HCl (CipHCl). The resultant membrane was found to inhibit both gram-positive bacteria (S. aureus, B. subtilis) and gram-negative bacteria (E. coli, S. typhimurium, V. vulnificus. The membrane was also biocompatible with good proliferation demonstrated by culturing 3T3-L1 fibroblasts. Chellamani et al (2014) showed that wound healing from electrospun polycaprolactone (PCL) loaded with tetracycline hydrochloride was faster than common wound dressing procedure. With 2% tetracycline hydrochloride loading into the electrospun membrane, bacterial reduction was 100% on S aureus and K. pnenmonial. In a further study by Ahire et al (2015) on ciprofloxacin (CIP) eluting nanofibers, blended poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO) nanofibers were used as the carrier material and tested against Pseudomonas aeruginosa and Staphylococcus aureus. Both bacteria are known to form biofilms and are commonly found in hospital acquired infections. All CIP was released from the PDLLA/PEO nanofibers within the first 3 hours. However, growth of P. aeruginosa continued to be inhibited for seven consecutive days although inhibition of S. aureus is only for 48 hours. Breast epithelial cells MCF-12A cultured on CIP loaded nanofibers were found to be viable thus demonstrating biocompatibility of the membrane.

A: Release of ciprofloxacin (CIP) from PDLLA: PEO nanofibers immediately after immersion into PBS, and 2, 3, 4, 6, 24 and 48 h thereafter, B: in vitro antimicrobial activity of CIP-containing nanofibers (CIP-F) against P. aeruginosa PA01 and S. aureus Xen 30, C: cell density of P. aeruginosa PA01 exposed to nanofibers without CIP (CF) and D: cell density of S. aureus Xen 30 exposed to CF and CIP-F. The control was without nanofibers and without CIP. Data points represent an average reading recorded from three disks per time point and three independent experiments (mean ± standard deviation). * p < 0.05. ns: not significant. [Ahire et al PLoS ONE 2015; 10: e0123648. This work is licensed under a Creative Commons Attribution 4.0 International.]

In the selection of anti-bacterial additives, natural and herbal extracts are sometimes preferred as they are perceived to be more biocompatible, non-toxic and reduced side effect. Motealleh et al (2014) loaded poly(e-caprolactone)/polystyrene blends with chamomile, extracted from chamomile plant, to give the resultant electrospun membrane anti-bacterial functionality. The chamomile loaded poly(e-caprolactone)/polystyrene composite fibers showed an initial burst release profile during the first 10 hours before gradually plateauing. The released chamomile was shown to be effective against S. aureuas bacteria and C. albicans fungi. In vitro studies using mesenchymal stem cell showed better proliferation on the drug loaded composite and in vivo study using a rat model also showed faster recovery on the drug loaded composite. Researchers have also constructed electrospun fibers that produce antibacterial chemicals in the right condition. Leonarta and Lee (2021) used electrospun polyvinyl alcohol (PVA) nanofibrous to separately encapsulate glucose oxidase (GOx) and glucose (Glu). In aqueous media, GOx would catalyze the reaction between glucose released from the PVA/Glu nanofibers and oxygen to produce hydrogen peroxide (H₂O₂). H₂O₂ is a strong oxidising agent which is able to kill bacteria. The mix of PVA/Glu nanofibers and PVA/GOx nanofibers were able to give a sustained release of H₂O₂ over 7 days in room temperature. Cross-linking of the nanofiber membranes using glutaraldehyde (GA) vapor prevent the nanofibrous membrane from dissolving in water and prolonged the release of H₂O₂ probably due to slower release of glucose from the nanofibers. The sustained release of H₂O₂ was found to be effective against both Escherichia coli and Staphylococcus aureus with Gram(+) S. aureus cells being more susceptible to H₂O₂ than Gram(-) E. coli and & gt;99% of S. aureus were killed after 1 h incubation with the membrane. Such mixture of nanofibers containing an enzyme and the biomolecules have the potential to be used for wound healing in particular diabetic patients which has a higher level of blood glucose for the production of H₂O₂.

Lysozyme is a natural occurring enzyme that is known to inhibit gram positive bacteria. As lysozyme is positively charged at neutral pH, Kehail and Brigham (2017) immobilized it on negatively charged electrospun poly(3-hydroxybutyrate-co-3-hydroxyhex-anoate) [P(HB-co-HHx)] fibers. The resultant electrospun fibrous composite scaffold was found to inhibited the biofilm formation (Rhodococcus opacus PD63) by 42% while solvent cast form was 30%. Better inhibition by electrospun fibers may be due to greater contact surface area and exposure of the bacteria to lysozyme.

Ponericin G1 is a natural antibacterial peptide extracted from ants and is known to be effective against fungi and bacteria but harmless to eukaryotic cells. Zhao et al (2019) used polydopamine (PDA) as bonding agent on electrospun poly(lactic-co-glycolic acid) (PLGA) fibers for surface adhesion of fibroblast growth factor (bFGF) and ponericin G1 for the construction of a novel wound dressing. The use of PDA was shown to improve loading efficiency of both bFGF and ponericin G1. The modified electrospun PLGA scaffold showed significant inhibition against S. aureus and E. coli. In vitro culture of BALB/C 3T3 cells showed good adhesion, proliferation and expression of tissue repair related genes. The presence of ponericin G1 does not have any impact on the cells when compared to scaffold without it. In vivo study using a Sprague-Dawley rat epidermal injury model showed excellent recovery especially with the groups containing bFGF with the same rate of recovery for scaffolds containing ponericin G1 and without.

Appropriate material selection may also allow electrospun wound dressing to be used in the long term and be made reusable. Ma et al (2011) showed that electrospun SiO₂ membrane loaded with silver nanoparticles can be reused with the same level of inhibition against E. coli after heat treatment at 380°C for 2 hours. Biocompatibility test using BMSCs (bone mesenchymal stem cells) showed good proliferation and cell viability with silver nanoparticle dosage less than 200µg/cm². With a relative low silver nanoparticle dosage of 13µg/cm², the membrane is already able to show 100% anti-bacterial efficiency.

In a recent study, the size of the fiber diameter was found to have some influence on bacteria adhesion and proliferation. When the fiber diameter is close to the size of the bacteria, proliferation was found to be the highest across the bacteria studied (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) [Abrigo et al 2015]. For rod shaped bacteria such as E. coli and P. aeruginosa, fiber diameters smaller than the bacterial length were found to induce cell death as it attempts to wrap round each fiber. However, the effect of fiber diameter on round S. aureus was less.

Bacteria test after 6 hours of incubation at 37°C: (a) pure EVOH nanofiber and the rest are nanofibers containing (b) gentamicin, (c) Ag nanoparticle, and (d) iodine [Wang et al. Journal of Nanomaterials, vol. 2015, Article ID 418932, 8 pages, 2015. This work is licensed under a Creative Commons Attribution 3.0 Unported License.].

The wide variety of active molecules that can be loaded into electrospun fibers allow the wound dressing to be tailored to its function. Hassiba et al (2017) created a bilayered composite of electrospun fibers with the top layer comprising of electrospun poly(vinyl alcohol) and chitosan loaded with silver nanoparticles (AgNPs) and a lower layer of polyethylene oxide (PEO) or polyvinylpyrrolidone (PVP) nanofibers loaded with chlorhexidine (an antiseptic). The top layer was meant to keep environmental germs and dirt from getting into the wound while the inner layer containing chlorhexidine was meant to facilitate wound healing. The bilayered electrospun composite membrane was found to inhibit Staphylococcus aureus, Escherichia coli , Pseudomonas aeruginosa and Candida albicans.

Bacteria trap

An interesting application of electrospun membrane is its potential ability to attract bacteria from the wound to the dressing. Abrigo et al (2015b) showed that surface modification of electrospun polystyrene with alkylamine was able to enhance attachment of E. coli. Conceptually, it may be able to draw bacteria from the wound and removed when the dressing is changed. However, more tests are needed to check the performance and viability of this strategy.

Application

Conventional application of wound dressing is to have the dressing packed in a sterile pouch. When there is a need to use the dressing, it is taken out of the pouch and applied directly onto the wound. Similarly, electrospun wound dressing may also be used in the same way. Palo et al (2019) constructed a bi-layered wound dressing with a solvent cast (SC) bottom layer and an upper layer consisting of either electrospun fibers or three-dimensional (3D) printing. Both layers were made from a blend of polyvinyl alcohol (PVA) and sodium alginate (SA). Adhesion tests showed that cross-linked electrospun layer significantly reduces adhesiveness compared to 3D printed macroporous layer and SC film. This is advantageous in wound dressing as it allows easy removable from damaged tissues. Their study showed that SC/3D printed dressing adhesive behaviour is similar to SC thus the 3D printed layer offers no advantage in terms of adhesion. Tests on cell biocompatibility using fibroblasts also showed no significant difference in the cell viability between SC/3D printed dressing and SC film. However, with the electrospun layer, cell viability was greater. Therefore, having an electrospun layer on SC film was shown to offer several advantages for wound dressing. Portable electrospinning devices may offer an alternative way of covering the wound. The device typically runs on batteries to generate the required high voltage for initiating electrospinning from a cartridge filled with the solution. Mouthuy et al (2015) described the details of their portable electrospinning device which have been shown to be capable of producing nanofibers from a wide range of polymers such as polycaprolactone, polyvinyl alcohol, polyethylene oxide and poly(vinyl butyral) to name a few. Their battery powered device is able to run for 100 minutes at 13 kV. Feasibility of the device in depositing fibers on skin was demonstrated on human volunteers and pig skin [Mouthuy et al 2015].

Portable electrospinning device. Photo credit: P-A Mouthuy, University of Oxford, UK

In a demonstration of wound coverage using in situ precise gas assisted electrospinning, Lv et al (2016) covered a simulated open head injury with electrospun N-octyl-2-cyanoacrylate (NOCA), a commercial tissue adhesive (medical glue), fibers. The NOCA nanofibrous membrane covering the hole showed no fluid leakage. A wound area of 4-9 cm² can be covered by a layer of nanofibrous membrane with 20s of electrospinning. The video below showed the wound coverage using electrospinning.

Liu et al tested the feasibility of using a hand-held portable electrospinning apparatus, HHE-1 from Qingdao Junada Technology Co., Ltd for deposition of fibers on a wound. This simple electrospinning apparatus ejects the solution through a syringe manually by the user's thumb. A disk covering the solution exit presumably to direct the electrospinning jet. Liu et al (2018) used polymers, poly(vinyl pyrrolidone) (PVP) and poly(vinyl butyral) (PVB) as the polymer carrier with iodine and iodine complex as anti-bacterial agents for electrospinning onto wound. PVP and PVB were both soluble in ethanol thus safe to use for direct application on wound. Comparison of the anti-bacterial properties of iodine-based electrospun membranes against E. coli and S. aureus showed that PVP with iodine had the best inhibition property and PVB/poly(vinylpyrrolidone)-iodine complex the least. The portable hand-held electrospinning device was able to deposit a layer of fibers on a hand and the fibers were sufficiently compact such that the deposited membrane may be peeled off from the hand.

The handheld electrospinning apparatus (a) and the in situ electrospinning process (b). The electrospinning jets can be seen from the spinneret [Liu et al 2018]

In a demonstration of the versatility of a handheld electrospinning device, Xu et al (2022) was able to deposit polyvinyl alcohol (PVA) fibers containing bone marrow-derived stem cells (BMSCs) onto full-thickness skin wounds. The PVA solution was prepared using phosphate-buffered saline (PBS) and 1?x?10⁷ BMSCs were added to the solution prior to electrospinning. Cell viability test using dead/live staining showed 90.15% survival rate immediately after electrospinning. In vivo tests using SD rats demonstrated significantly faster wound healing in the PVA/cell group compared to PVA only and untreated control. On day 14, the wound covered with the PVA/cell group was almost completely closed. The epidermis was completely covered by epithelial tissue with normal skin appendages found around the wound. Healing was slower in the PVA only group with the subcutaneous tissues thinner than the PVA/cell group. The group without any intervention were still in the granulation tissue repair state with many capillaries, fibroblasts and inflammatory cells.

One main concern about direct electrospinning application onto wounds is the presence of solvents. If electrospinning is carried out by melt polymer, there will be no solvent and it can be safely applied onto the wound. For a portable melt electrospinning device, the challenge is to insulate the heating element from the high voltage. In a laboratory setup, it is possible to physically isolate the heating element from the high voltage. In a portable device, the space constraints means that both parts will be at close proximity to one another. A unique material which has excellent heat conductivity but electrically insulating property is the key to construct a portable melt electrospinning device. This allows the heat to be conducted close to the nozzle tip where the high voltage is applied while insulating the heating element from the high voltage source. Aluminum nitride (AlN) is one such material with good electrical insulation and heat transfer capacity. Zhao et al (2020) showed that with an AlN tube to conduct the heat within the setup, they were able to melt electrospin poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and hot-melt adhesive. The device was tested on a mouse for direct fiber deposition on a cut wound. The PCL fiber mesh that was melt electrospun on the wound was able to prevent blood from spilling out.

Optical pictures showing the process of melt e-spinning PCL fibers directly onto the skin producing by the hand-held melt e-spinning apparatus in 5 min and SEM images of fibers with various polymer materials produced by the hand-held melt e-spinning apparatus to further test the performances of the apparatus. (a) The apparatus was operated by one hand and the other hand receives the PCL fibers. (b) Magnified view of spinning jet. (c) Comparison of two hands with or without fiber membrane. (d) The picture shows the e-spun fiber membrane has good flexibility and the inset SEM picture is the e-spun fibers. e PLA fibers. f PLGA fibers. g hot-melt adhesive fibers [Zhao et al 2020].

Multi-functionality

The versatility of electrospinning means that the resultant fibers can exhibit multiple functionality to aid wound healing. Core-shell fibers can be easily constructed such that the core and sheath material have different role to play in the wound dressing. Zhan et al (2022) constructed multifunctional core-shell electrospun fibers for the purpose of diabetic wound healing. Four different materials were used in the fibers. Chitosan (CS) which exhibits antibacterial activity was used as the shell material so that the dressing would inhibit bacteria found on the wound. The core contains bioactive compounds, copper (Cu) salt and decellularized Wharton's jelly matrix (DWJM), and poly(L-Lactide-co-caprolactone) (PLCL). Cu is known to promote angiogenesis and endothelial migration and this will help wound healing at the intermediate stage. DWJM contains a natural extracellular matrix (ECM) and a source of endogenous growth factors to promote collagen deposition and wound healing. PLCL was added into the core matrix to provide stable electrospinning and mechanical strength for the composite fiber. In vivo test carried out using Sprague-Dawley (SD) rats diabetic model showed that the CTS/PLCL/DWJM/Cu core-shell nanofibers wound dressing exhibited the fastest healing compared to other wound dressings with one or more active substances absent. Skin appendages including hair follicles were evident at day 16 for the CTS/PLCL/DWJM/Cu core-shell nanofibers wound dressing group while healing for the other groups were slower.

Multiple spinnerets with each spinning a material with a particular functionality may be used to form a mat with a mixture of fibers. Du et al (2017) used a dual spinnerets electrospinning setup to form a membrane with antibacterial and wound healing properties. In their setup, electrospun fibers were made out of poly(vinyl alcohol) (PVA)/silver nanoparticles (AgNPs) and poly(caprolactone) (PCL)/ascorbyl palmitate (AP). The silver nanoparticles (AgNPs) are the antibacterial agent while AP aids wound healing. In vitro cytotoxicity test using 3T3 fibroblasts showed that proliferation were reduced with electrospun PVA/PCL/AgNPs compared to neat PVA/PCL. With PVA/PCL/AgNPs/AP, the fibroblast proliferation was 87% of the control which demonstrates the benefit of the presence of AP. In vivo studies using a rat model showed that wound closure from PVA/PCL/AgNPs/AP electrospun fibrous mat has the best result compared to all other groups including PVA/PCL/AgNPs, PVA/PCL/AP and PVA/PCL mesh. Li et al (2018) created a multi-functional wound dressing by creating a double layer electrospun membrane. The inner layer that comes into contact with skin was made of electrospun chitosan for its biocompatibility and and intrinsic antibacterial nature. Electrospun polycaprolactone (PCL) fibers were used as the outer layer to instill mechanical strength to the membrane. Anti-bacterial compounds, lidocaine hydrochloride (LID) and mupirocin was added to chitosan and PCL fibers respectively. The release profiles of LID and mupirocin were very different. 66% of LID was released in the first hours followed by gradual release to 85% in the following 6 hrs. Mupirocin showed an initial release of 57% in the first 6 hrs followed by sustained release of another 30% over the next 5 days. Therefore it can be seen that LID was released very quickly during the initial phase while mupirocin was able to maintain a gradual release for the remaining half of its load after the first 6 hrs. Slower release of mupirocin was attributed to stronger chemical linkage with PCL molecules. Such drug release profile is clinically relevant to achieve therapeutic concentration of the drug in minimal time. Schulte-Werning et al (2021) used blending to incorporate several functionalities into the electrospun fibers. The ingredients forming the blend were chloramphenicol (CAM), an antibiotic, water-soluble β-1.3/1.6 glucan (SBG®), an active ingredient in the topical treatment of diabetic foot and leg ulcers, chitosan (CHI), a bioactive polymer which exhibits intrinsic antimicrobial activity, hydroxypropylmethylcellulose (HPMC), a cellulose-based polymer with high swelling capacity, and polyethylene oxide (PEO) to facilitate fiber formation from electrospinning. Instead of using conventional nozzle electrospinning, an Elmarco NanospiderTM NS Lab machine with electrode wire as the spinneret. The resultant electrospun multi-functional nanofibers were shown to inhibit E. coli and S. aureus, non-toxicity towards keratinocytes (HaCaT cells) and macrophages (RAW 264.7), and exhibit anti-inflammatory activity. Interestingly, in the sample that contains CHI but not CAM, no antimicrobial effect was seen and this has been attributed to the neutral pH of the gel. CHI requires an acidic environment for protonation so as to exhibit antimicrobial effect. Although CHI does not contribute to antimicrobial functionality in this wound dressing, its presence increases the mechanical strength and improves the anti-inflammatory of the membrane in the presence of SBG®. Upon exposure to fluid, this multi-functional nanofiber membrane showed a high swelling index and became transparent. This helps to adhere to the wound and maintain a moist local environment. High transparency of the membrane also allows examination of the wound without the need to remove the dressing.

A wound dressing may be constructed from different materials or processes to take advantage of the properties provided by each structure. Liu et al (2023) constructed a skin substitute using multi-layers of electrospun polycaprolactone (PCL) fibers and sprayed alginate hydrogel powder by alternating electrospinning and spraying. The multi-layered structure was wetted and cross-linked so that the alginate formed a stable matrix with the PCL fibrous layers to form a fiber hydrogel interpenetrated network (FHIPN). Amino-terminated hyperbranched polyamide (ATHBP) with antibacterial properties was added to the FHIPN by dipping the FHIPN in ATHBP solution to form functionalized fiber-hydrogel interpenetrating network (FFHIPN). The amino groups on ATHBP would bind to the carboxyl groups of sodium alginate through Coulomb interaction. In this setup, the PCL fibrous layers would provide the necessary elasticity and mechanical strength. On a wound injury, the alginate hydrogel would absorb the wound exudate and swell to create a more open FHIPN structure which encourages cell infiltration and at the same time provides a wet environment for wound healing. In vivo study using mouse full-thickness wound defect model showed significantly better wound healing using FHIPN and FFHIPN compared to the negative control using Gauze. In particular, histological tests showed the appearance of hair follicle structures on FHIPN and FFHIPN treated mice by day 7 while a small amount of hair follicle appeared in the wounds treated with Gauze on day 14. At day 14 there was almost complete wound closure for FFHIPN treated wounds.

In vivo

In vivo studies are critical to examine the efficacy of the device in the complex physiological environment. It is also an opportunity to test the actual application of the device. Shabunin et al (2019) tested the feasibility and efficacy of a bilayer electrospun dressing for treatment of burnt wound. The two electrospun layers comprised of a non-biodegradable alcohol-soluble aliphatic copolyamide (CoPA) (copolymer of poly(ε-caprolactam) [-NH-(CH₂)₅-CO-]n and poly(hexamethylenediamineadipinate) [-NH(CH₂)₆NHCO(CH₂)₄CO-]n) layer to provide mechanical support and a degradable chitosan/chitin layer to facilitate wound healing. The CoPA electrospun layer is separated when the dressing is removed leaving behind the chitin/chitosan layer to facilitate the epithelialization of the wound. In their In vivo study using third degree burn model on rats, almost complete (up to 97.8%) epithelialization of the wound surface had been achieved within 28 days for the group with the bilayer electrospun dressing. There were no deaths or purulent complications in the target group while there are 25% and 59.7% cases respectively in the control group. With electrospun CoPA membrane only, there were no deaths but 11% purulent complications. The bilayer electrospun wound dressing have shown the potential for treatment of burn wounds.

Another area of investigation is the type of active compounds added to the scaffold to promote wound healing. Bioactive compounds such as growth factors and others are often incorporated into electrospun wound dressings to accelerate the healing process. Losi et al (2020) compared the efficacy of wound healing using electrospun fibrin-based scaffold loaded with platelet lysate (PL). PL is derived from freeze-thawing cycles of platelet concentrates from peripheral blood and this contains a complex composition of growth factors and other biomolecules. Tests on a full thickness skin wound diabetic mice showed that fibrin-based scaffold loaded with PL performed better than electrospun scaffold loaded with growth factors, neat electrospun scaffold and Mepore® after 14 days. In the construction of the electrospun fibrin-based scaffold, fibrinogen solutions were first used to electrospun into fibers. Thrombin was subsequently sprayed onto the fibrinogen fibers and incubated for 30 min to allow fibrin polymerization. This method of preparing fibrin fibers allows bioactive compounds to be loaded as there are no post-electrospinning washing or chemical cross-linking steps.

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