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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process a
b
c
Aysu Arslan , Murat Şimşek , Sevcan Dalkıranoğlu Aldemir , Nur d
Merve Kazaroğlu & Menemşe Gümüşderelioğlu
abd
a
Department of Chemical Engineering, Hacettepe University, Ankara 06800, Turkey b
Department of Nanotechnology and Nanomedicine, Hacettepe University, Ankara 06800, Turkey c
Department of Biology, Hacettepe University, Ankara 06800, Turkey d
Department of Bioengineering, Hacettepe University, Ankara 06800, Turkey Published online: 20 May 2014. To cite this article: Aysu Arslan, Murat Şimşek, Sevcan Dalkıranoğlu Aldemir, Nur Merve Kazaroğlu & Menemşe Gümüşderelioğlu (2014) Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process, Journal of Biomaterials Science, Polymer Edition, 25:10, 999-1012, DOI: 10.1080/09205063.2014.918455 To link to this article: http://dx.doi.org/10.1080/09205063.2014.918455
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 10, 999–1012, http://dx.doi.org/10.1080/09205063.2014.918455
Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process
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Aysu Arslana, Murat Şimşekb, Sevcan Dalkıranoğlu Aldemirc, Nur Merve Kazaroğlud and Menemşe Gümüşderelioğlua,b,d* a Department of Chemical Engineering, Hacettepe University, Ankara 06800, Turkey; Department of Nanotechnology and Nanomedicine, Hacettepe University, Ankara 06800, Turkey; cDepartment of Biology, Hacettepe University, Ankara 06800, Turkey; dDepartment of Bioengineering, Hacettepe University, Ankara 06800, Turkey b
(Received 23 December 2013; accepted 23 April 2014) In this study, fibrous mats were fabricated via electrospinning from solutions of polyethylene terephthalate (PET), PET/chitosan, and PET/honey at different concentrations. The effect of honey and chitosan on electrospinning process was investigated and compared. Fibers containing chitosan had a beaded or ribbon-like/ branched morphology, but this morphology improved in the presence of honey. The diameter of electrospun fibers decreased with an increased ratio of honey in PET solution. In addition, fiber deposition area in the collector increased by increasing the honey content. PET/chitosan and PET/honey fibrous mats reached an equilibrium water content in 15 min and their water uptake capacities, which are important for exudating wounds, were found in the range of 280–430% on dry basis. Cytotoxicity evaluation demonstrated that fibers exhibited no cytotoxic activity. This study discloses that PET fibrous mats especially electrospun in the presence of honey could be proposed as potential wound dressing materials owing to their improved processing abilities besides their suitable structural properties. Keywords: electrospinning; fibers; polyethylene terephthalate; chitosan; honey; wound dressing
1. Introduction Since the Egyptian times, a suitable material has been required to cover a wound for preventing any infection and thus for performing effective wound healing. Throughout history, honey pastes, plant fibers, and animal fats had been used as wound dressings.[1] Wound dressings are usually used to encourage the various stages of wound healing by creating better healing conditions.[2,3] An ideal wound dressing material is expected to have the following properties: (i) maintaining the most suitable environment at the wound/dressing interface, (ii) absorbing excess exudates, (iii) providing thermal insulation, (iv) providing mechanical and bacterial protection, (v) allowing gaseous and fluid exchanges, (vi) to be non-allergic, and non-scarring.[1,4] The wound-dressing materials produced by the electrospinning method have excellent properties when they were compared with the dressings obtained by conventional methods, including the preparation of polymeric films and foams.[2,5] Nanofibrous wound dressings having small holes and high effective surface area can promote *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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hemostasis phase.[1] Due to the high surface area to volume ratio of the nanofibers, they exhibit good water absorption capacity. Thus, if hydrophilic polymer fibers are employed, the fibrous dressings have a capacity to absorb wound exudates more efficiently than the conventional ones. Also, bacterial infection can be effectively inhibited due to the small pore size. Electrospun fibrous wound dressings can also meet the requirement of high gas permeation.[1] Various synthetic and natural polymers or their combinations have potential usage as electrospun nanofibrous wound dressings.[6] Among them, polycaprolactone (PCL), poly (L-lactic acid), polyvinyl alcohol, chitosan, and gelatin have been widely used.[7,8] Additionaly, in order to obtain electropun mats with desirable antibacterial characteristics two approaches have been used: (i) immobilization of drugs or antibacterial agents into the electrospun nanofibers and (ii) electrospinning of polymers which have intrinsic antibacterial and wound-healing properties, e.g. chitosan.[8] Polyethylene teraphthalate (PET) is a semicrystalline polyester that is widely used in the production of vascular grafts, films, fibers, containers, and engineering plastics. The properties of polyesters also make them potential candidates for producing fibers. Electrospun PET nanofibers were first obtained by Kim et al. [9] and nowadays, it has been used in electrospinning process to obtain nano/micro fibers especially for tissue engineering,[8,10] due to its advantageous mechanical properties, biocompatibility, and cost-effectiveness. Chitosan has excellent properties such as biodegradability, biocompatibility, and antibacterial and wound healing activity.[11–14] Recently, electrospun fibers based on chitosan have been widely investigated owing to their potential applications in various areas.[7,15,16] However, some difficulties may arise from the use of chitosan because its solutions could not be easily processed. Jung et al. [7] fabricated PET/chitosan electrospun fibrous matrices and they determined that these matrices had an antibacterial effect and biocompatibility. Honey is generally composed of glucose (30%), fructose (40%), sucrose (5%), water (20%), and many other substances, such as amino acids, vitamins, minerals, and enzymes.[17] It has been used in wound care, due to its medicinal properties. It is known that honey can provide a moist healing environment without the risk of bacterial infection.[18] Honey also has an antimicrobial property.[19,20] Maleki et al. [17] showed that fabrication of the electrospun honey-based fibrous matrices could be possible. They examined poly(vinyl alcohol) (PVA)/honey electrospun fibers and obtained uniform, bead-free fibers. In this study, PET, PET/chitosan, PET/honey, and PET/chitosan/honey solutions at different weight ratios were electrospun to obtain potential wound-dressing materials. The effect of chitosan and honey on fiber formation and morphology was compared. The resulting fibrous mats were well characterized in view of wettability, water-content, chemistry, and also cytotoxicity. 2. Materials and methods 2.1. Materials Chitosan (85% deacetylated, medium molecular weight) was supplied from SigmaAldrich. Trifluoroacetic acid (TFA) as a solvent for electrospinning was purchased from Merck. Commercial PET (MW: 49,500 g/mole) and honey from flowers in Meydancık country side of Artvin province in Turkey were used in the study.
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For cell-culture studies, 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) and penicillin-streptomycin solution were obtained from Sigma-Aldrich (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and trypsin/EDTA solution were purchased from Hyclone (US).
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2.2. Electrospinning of PET-based solutions Each electrospinning solution consisting of PET or PET/chitosan at certain weight ratios given in Table 1 was dissolved in TFA and stirred overnight at room temperature. For PET/honey solution (in TFA), complete dissolution time of PET increased up to 48 h with the increasing honey concentration from 10% to 40% (wt). Spinning solutions were transferred to a 2.5 mL syringe with a 21-gauge needle. Then, a high voltage (Gamma High Voltage Research, Ormond Beach, FL) was applied between the needle placed into syringe pump (NE 300, New Era Pump Systems) and stationary collector. Fibers were collected on an aluminium foil fixed onto the collector for a time period from 20 to 60 min to obtain approx. 140-μm thick fibrous matrices. 2.3. Characterization of fibrous mats Morphology of electrospun fiber mats was observed via a scanning electron microscope (SEM, Zeiss Evo 50, Germany). Fiber diameters were calculated using ImageJ software (NIH, Bethesda, MD) by taking the average of at least 100 measurements from SEM images. Attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) was performed to determine the presence of chitosan and honey in PET structure by using a Thermo Scientific Nicolet iS10 FTIR spectrophotometer (USA) bearing an ATR attachment with a diamond crystal. The water contact angles of electrospun fiber mats were measured by sessile drop method at room temperature (Kruss DSA 100, Germany) immediately after the deionized water droplet made contact with the fiber surface. Dynamic water uptake studies were carried out to determine equilibrium water contents of fiber mats. Experiments were done in excess phosphate buffer saline (PBS, pH: 7.4, 37 °C) for selected time intervals. The fibrous mats were soaked in PBS until
Table 1. Polymer concentrations and compositions used for the production of fibrous mats and their abbreviations. Final polymer concentration (%) 17
19
Compositions in 100 mL 15.3 mg PET + 1.7 mg chitosan 15.3 mg PET + 1.7 mg chitosan + 1.7 mg honey 17.0 mg PET + 1.7 mg honey 19.0 mg PET 17.1 mg PET + 1.9 mg chitosan 19.0 mg PET + 1.9 mg honey 19.0 mg PET + 3.8 mg honey 19.0 mg PET + 5.7 mg honey 19.0 mg PET + 7.6 mg honey
Abbreviations of fibrous mats 17(PETCs10) 17(PETCs10Hny10) 17(PETHny10) 19PET 19(PETCs10) 19(PETHny10) 19(PETHny20) 19(PETHny30) 19(PETHny40)
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they reached maximum water contents. Fibrous mats were removed from the swelling medium with certain time intervals, the excess liquid drops on the surface were wiped with a blotting paper and the weight of each mat was measured. The measurements were continued until the weight of the swollen mats reached a maximum value. Triplicate data were obtained for each measurement. The following equation (Equation 1) was used to calculate the mass swelling ratio based on dry weight.
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Q ¼ ½ðWs Wd Þ=Wd 100
(1)
where Ws is the weight of swollen sample and Wd is the weight of dried sample. The porosities of electrospun fiber mats were calculated using ImageJ software developed by the National Institutes of Health using ‘Auto Local Threshold’ plugin. The analysis was carried out via SEM images of electrospun mats having a similar thickness. 2.4. Cytotoxicity tests 2.4.1. Preparation of liquid extracts of mats The cytotoxicity of the electrospun fibrous mats was evaluated based on a procedure adapted from the ISO10993-5 standard test (MEM-extract test/MTT test system-72 h incubation) method. Experiments were carried out with L929 mouse fibroblasts obtained from HUKUK Cell Line Collection (No. 92123004, Foot and Mouth Disease Institute, Ankara, Turkey). PET, PET/chitosan, and PET/honey fibrous mats were compared in terms of cellular toxicity. Fiber mats were sterilized by ultraviolet (UV) light for 30 min. In this study 33 cm2 of sterilized PET, PET/chitosan and PET/honey fiber mats were extracted at 37 °C for 24 h in 35 mL culture medium. The medium consisted of DMEM with 10% (v/v) FBS, 1% penicillin-streptomycin, and 2.5% L-glutamine. After 24 h of incubation, fiber mats were removed from the medium and the extract was filtered using 0.22 μm filter (Orange Scientific). 2.4.2. Cell seeding and extract treatment Cell cultures were conducted in sterile 24-well tissue culture polystyrene (TCPS) dishes in stationary conditions. During the extract preparation, L929 cells were seeded into 24-well plates at an inoculation density of 5 × 104 or 6 × 104 cells/mL. After a 24 h incubation period the culture medium was replaced with various dilutions of the extract (25, 50, and 100%) to determine the cytotoxicity of fibrous mats by MTT assay and crystal violet staining. 2.4.3. Cell viability and growth kinetics of L929 cells Following 1st, 2nd and 3rd days of culture, the medium was removed and 600 μL prewarmed serum-free DMEM and 60 μL MTT solution (2.5 mg/mL dissolved in PBS) were added to each well which was incubated at 37 °C for 3 h. Then, the medium was removed from each well and 0.04 M HCl containing 400 μL isopropanol solution was added to dissolve formazan crystals. After pipetting the solution, violet colored 200 μL supernatant was transferred to 96-well plate and its optical density was measured spectrophotometrically at 570 nm with reference to 690 nm using a microplate reader (Asys UVM 340, Austria). Simultaneously, the morphology of the cells was observed
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by crystal violet staining. After 24 h incubation with the extract, the medium was removed from each well and washed two times with PBS. The cells were fixed in acetone-methanol solution (1:1) for 20 min. Then, the solution was removed and each well was treated with crystal violet solution (0.05% lyophilized crystal violet in 20% methanol) for 30 min. Each well was washed properly and examined under the inverted microscope (Olympus, USA).
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3. Results and discussion 3.1. Characterization of PET-based fibers Electrospinning conditions were optimized to obtain smooth and continuous fibers. For obtaining desirable fibrous mats, polymer concentration is a key parameter. For this reason, while the polymer (PET or PET/chitosan) concentration was varied from 15% (w/v) to 20% (w/v) to obtain smooth and continuous fibers, optimum process conditions were determined by changing the flow rate between 0.5 and 4.0 mL/h, applied voltage between 15 and 27 kV, and fiber collecting distance between 15 and 30 cm. The results revealed that discontinuous, coiled, or beaded fibers formed below 19% of polymer wt ratio. Above this concentration, electrospinning failed due to high viscosity, which is making the solution difficult to be ejected from a syringe. It was also observed that branched jet formation occured at a lower flow rate of 3 mL/h due to the formation of secondary jets. These jets could not reach the collector and thus it was difficult to obtain a stable Taylor cone which is essential for fiber production. The experiments showed that the following electrospinning parameters enabled best results in terms of fabrication of fibrous mats to be used: 19% (w/v) of polymer concentration, 3.5 mL/h of flow rate, 24 kV of applied voltage, and 35 cm of collecting distance. In this study, 17% (w/v) of PET/chitosan and PET/chitosan-honey solution were also used to observe the improvements in fiber morphology/production in the presence of honey. Table 1 shows polymer concentrations and compositions of solutions used for the production of fibrous mats with their abbreviations. Basic properties of fibrous mats are presented in Table 2. In order to investigate the effect of honey on the spinnability of PET/chitosan fibers, 10% (wt) of honey was added to the solution of 17(PETCs10) for preparing 17(PETCs10Hny10) matrix. Morphological differences between 17(PETCs10)
Table 2. Basic properties of fibrous mats (thickness: approx. 140 μm) prepared from different PET, chitosan, and honey compositions at the optimized electrospinning condition (voltage: 24 kV; flow rate: 3.5 mL/h; distance to collector: 30 cm). Composition of fibers 17(PETCs10) 17(PETCs10Hny10) 17(PETHny10) 19PET 19(PETCs10) 19(PETHny10) 19(PETHny20) 19(PETHny30) 19(PETHny40)
Fiber (nm)
Matrix porosity (%)
Equilibrium water content (%)
Water content angle (°)
565 + 245 557 + 217 678 + 145 682 + 111 1484 + 389 780 + 106 755 + 128 720 + 130 668 + 177
50.54 47.27 49.36 47.86 43.39 44.21 45.39 49.03 49.07
nd nd nd 430 + 20 426 + 21 418 + 27 nd nd 282 + 9
nd nd nd 135.0 + 1.5 0 134.8 + 1.3 nd nd 132.7 + 2.1
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Figure 1. SEM images of PET, PET/chitosan (PETCs) fibrous mats, and their honey-including forms (PETHny and PETCsHny, respectively) in different forms. (a) 17(PETCs10) (1KX), (b) 17 (PETCs10Hny10) (2KX), (c) 17(PETHny10) (2KX), (d) 19PET, (e) 19(PETCs10) (1KX), and (f) 19(PETHny40) (2KX). The photographs show higher magnifications.
and 17(PETCs10Hny10) fiber mats can be clearly seen from Figure 1(a) and (b). While the 17(PETCs10) fibers having a beaded morphology, the stability of the jet was enhanced in favor of the formation of bead-free fibers with the addition of honey. Since higher amount of solvent molecules in 17(PETCs10) than that of 19(PETCs10) caused an increase in the surface tension of polymer solution at this concentration (17% w/v), bead formation along with fiber structure appeared because of insufficent chain entanglement. However, no significant change in their fiber diameters was observed (Table 2). Figure 1(c) shows the SEM image of 17(PETHny10) fibers. Unlike 17(PETCs10) fibers, smooth 17(PETHny10) fibers were obtained in the presence of honey.
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Figure 1(d) shows that 19PET fibers have smooth and regular morphology with a diameter of 682 ± 111 nm. Veleirinho et al. [21] produced continuous nanofiber structures from 10% to 30% (w/v) PET solution by changing solvents (dichloromethane and TFA) and their volume ratios. They reported that fibers spinned from 30% w/v of PETTFA solution exhibited irregular morphology with branches or twigs. In this study, we used TFA as a solvent; however, in our optimized conditions, we did not observe similar defects related to PET fibers. In order to investigate the effect of chitosan on fiber morphology, 19% (w/v) of PET/chitosan solution (19(PETCs10)) was prepared. 19(PETCs10) fibers having 1484 ± 389 nm of diameter had partly ribbon-like and branched morphology when compared to 19PET fibers (Figure 1(e)). Also, it was seen that relatively very thin fibers arose from secondary jets separating main jet existed through the mat and they caused an increase in heterogenity of fiber diameter. Although polymer concentrations of 19PET and 19(PETCs10) were equal to each other, the fiber diameters increased in the presence of chitosan. This can be attributed to the polycationic structure of chitosan which has a significant effect on decreasing bending and stretching of the jet due to viscoelastic forces dominating the Columbic forces.[22–24] Similar results were reported in the relevant literature. Min et al. [25] stated that continuous fiber formation was inhibitied by repulsive forces between ionic groups within chitosan backbone under a high electric field. Ignatova et al. [8] reported that the electrospinning of poly[(L-lactide)-co-(D,Llactide)] (PLA) solution led to the formation of defect-free fibers, while the electrospinning of PLA/chitosan was causing broader distribution of fiber diameters than that of PLA fibers. In addition, in the case of PLA/chitosan systems splitting and branching of the fibers sometimes occurred. They explained that the elongation of the jet and the evaporation of the solvent could cause some shift in the balance between the surface tension and electrical forces and could result in jet splitting or branching of the fibers. In order to enhance the spinnability of chitosan and to obtain smooth fibers, some agents can be used as surfactants. Kriegel et al. [26] prepared PEO/chitosan fibers in the presence of surfactant molecule, sodium dodecyl sulphate, and reported that spinnability of jet was enhanced and smooth fibers were obtained. In order to examine the effect of honey on PET fibers, 19% (w/v) of PET/honey fibers were produced by increasing the honey ratio from 10 to 40% wt in the PET solution (Table 1). At a higher honey ratio than 40% wt, it was difficult to dissolve PET, completely. PET/honey fibers exhibited smooth and uniform fiber formation similar to PET fibers (Figure 1(f)). Maleki et al. [17] also produced uniform and smooth PVA/ honey nanofibers, independent of the honey content. In our study, fiber diameters slightly decreased with increasing honey content in spinning solution (Table 2). A decrease in fiber diameter can be explained with solution conductivity. If conductivity of a solution increases, more charges will be carried by the electrospinning jet. An increase in charge density results in a greater bending instability and stretching forces hence, fiber diameter decreases.[22] Since the conductivity of honey (90–130 × 10−5 S cm−1) is higher than that of chitosan (10−10 to 10−9 S cm−1),[27–29] a decrease in diameter of 19(PETHny10) fibers was observed. Fiber deposition area was also strongly affected by the honey ratio, inducing greater bending instability and stretching forces. 19(PETHny10) and 19(PETHny40) fibers had a deposition area of about 217 and 380 cm2, respectively, while the area was about 56 cm2 for 19(PETCs10) fibers. Therefore, collection time was increased from 20 to 60 min for obtaining fibrous mats having 140 ± 8 μm of thickness. Results showed that honey had a considerable effect on improving fiber production and fabrication during electrospinning process.
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Porosities of fiber mats are listed in Table 2. It can be seen that fiber porosities are almost the same, approx. 50%.
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3.2. ATR-FTIR analysis In ATR-FTIR spectrum of 19(PETCs10) matrix (Figure 2), O bridge stretching of the glucosamine residue and asymmetrical C–H bending of the CH2 group which belongs to chitosan were observed at 1042 cm−1 and 1371 cm−1, respectively. Figure 3 shows ATR-FTIR spectra of PET pellets, honey, and 19(PETHny40) mats. Since honey consists of mainly hydrated glucose and fructose, together with higher carbohydrates in smaller amounts, typical IR spectrum of honey is dominated by the features related to hydroxyl groups of carbohydrates, along with the bands due to skeletal vibrations of carbohydrates. The absorption of less abundant honey components is mainly overlapped. Compared to PET pellets, 19(PETHny40) fibers have additional characteristic peaks including C–H stretching at 2963 cm−1 (carboxylic acids and NH3+ of free amino acids), O–H stretching at 3420 cm−1 (water and carbohydrate), and other characteristic peaks (600–1500 cm−1) of carbohydrates, which indicates the presence of honey. 3.3. Wettability and water contents of fibrous mats Water contact angles of fiber mats were measured immediately after the water droplet made contact with the fiber surface. Table 2 gives the water contact angles of 19PET, 19(PETCs10), 19(PETHny10), and 19(PETHny40) fiber mats. While the contact angles of PET and PET/honey fibers are almost same, the contact angle of chitosan-blended mat, i.e. 19(PETCs10) mat, was found to be zero because of strong water affinity of chitosan. Fluid affinity is a very important parameter in terms of fluid absorption of a
Figure 2.
ATR-FTIR spectra of PET pellets, chitosan flakes, and 19(PETCs10) fibrous mats.
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ATR-FTIR spectra of PET pellets, honey, and 19(PETHny40) fibrous mats.
wound-dressing material especially for exudates wounds.[1] In a study,[30] although PET nanofibers had about 128° of the water contact angle, they became a highly wettable material (0°) after surface modification either by poly(methacrylic acid) grafting or by gelatin grafting. Equilibrium water contents of fibrous mats are given in Table 2. Although PET and PET/honey fibers exhibited hydrophobic behavior at the time of water contact with fiber surface, they absorbed water in a swelling medium. This behavior can be attributed to the porous structure of the fibrous mats allowing water access. All mats except 19PET swelled rapidly in 15 min. 19(PETCs10) and 19(PETHny40) fibers reached a maximum water uptake capacity at that period, whereas it took 180 and 90 min for 19PET and 19(PETHny10) fibers, respectively. Although conventional film dressings only show water absorption of 2.3%,[1] our PET-based fiber mats exhibited the absorption up to 280%. Xu et al. [31] prepared wound-dressing materials using chitosan/hyaluronic acid and examined their water uptake capacity. They found that the dressing materials had water content in the range of 200–450% in dry basis.
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3.4. Cytotoxicity evaluation In order to find out if the fibrous mats release toxic products or not, cytotoxicity test was carried out. The cytotoxicities of PET, PET/chitosan and PET/honey fibrous mats were determined by testing the viabilities of L929 cells through the colorimetric estimation using MTT assay. In this test, after exposure to the various dilutions of extract (i.e. 25, 50, and 100%), proliferation of L929 cells was measured for 3 days. As a control, no extract-added cell cultures were used. Absorbance values are evaluated as the measure of cellular proliferation. The extracts of the mats showed no inhibition of cell metabolism compared to the control group (Figure 4). Mitochondrial activities of L929 cells cultured with mat extracts have no statistically significant difference from control groups although the average absorbance values were less than that of the control group. In addition, cell death and morphological damage were not observed throughout the incubation period. Figures 5 and 6 present the crystal violet images of L929 cells at the first and second days of culture at different concentrations of PET, PET/chitosan, and PET/honey extract media. The number of cells has increased with time as expected. However, cells that are cultured with extracts of PET/honey (10% and 40% wt of honey) mats are more spindle-shaped and less confluent than other groups at the first day of culture period. As seen from Figure 5, these cells have elongated morphology when compared with cells incubated in PET and 19(PETCs10) extract groups. On the second day of
Figure 4. Mitochondrial activities of L929 fibroblasts proliferated in the presence of 0, 25, 50, and 100% extracts of the fibrous mats. (a) 19PET, (b) 19(PETCs10), (c) 19(PETHny10), (d) 19(PETHny40). Statistically significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001).
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Figure 5. First day crystal violet images of L929 fibroblasts proliferated in the presence of 0, 25, 50, and 100% extracts of the fibrous mats. (a–d) 19PET, (e–h) 19(PETCs10), (i−l) 19(PETHny10), and (m–p) 19(PETHny40) (magnifications are ×40).
culture (Figure 6), the group treated with extract of 19(PETHny10) started to gain normal morphology similar to PET, 19(PETCs10) extract groups, and control groups. On the other hand, the cells treated with the extract of 19(PETHny40) mats continued to protect their spindle-shaped morphology. However, at the end of the third day these cells also gained normal morphology (photographs were not given). These type of morphological changes arising from honey had been previously reported on the human keratinocyte cells that showed distinct cytoplasmic projections.[32] Additionally, Barui et al. [33] successfully investigated the role of honey on the expression of Type I and Type III collagens for 3T3 fibroblast cells and E-Cadherin for HaCaT cells during the cell–cell and cell–material interactions on the honey-alginate fibrous mat. They prepared fibrous honey-alginate mat via wet-spinning technique through physical entrapment of honey into alginate fibers. In the presence of honey, higher cellular viability and maturation have been observed on the alginate structure. Immuno-cytochemical assays demonstrated higher expression of collagen I and collagen III on the honey-alginate mat. Honey-including alginate scaffold induced to gain of functional dimensions
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Figure 6. Second day crystal violet images of L929 fibroblasts proliferated in the presence of 0, 25, 50, and 100% extracts of the fibrous mats. (a–d) 19PET, (e–h) 19(PETCs10), (i–l) 19(PETHny10), and (m–p) 19(PETHny40) (magnifications are ×40).
on 3T3 fibroblasts, as dependent on collagen synthesis. On the other hand, enhanced membranous expressions of E-cadherin in HaCaT cells on the honey-based materials demonstrated better impact in establishing cell–cell adhesion in the state of confluence. When considered together with the results of cytotoxicity assay and morphological imaging, electrospun PET/honey mats have the potential to be used as a wound-closure material. 4. Conclusions In the presented study, as potential wound-dressing candidates PET, PET/chitosan, and PET/honey fibers were electrospun and their physical structures were examined. Smooth and uniform PET/honey fibers were obtained up to 40 wt% honey. In the case of PET/chitosan, while the fibers had a beaded morphology at a lower polymer concentration (17% w/v), fiber diameter increased and ribbon-like/branched fibers were observed at a higher polymer concentration (19% w/v). The jet stability was improved
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and processing limitations related to the spinning of chitosan were eliminated with the addition of honey to the spinning solution. All fiber mats showed good water-absorbing capacity when compared to typical wound dressings. MTT results demonstrated that electrospun PET, PET/chitosan, and PET/honey matrices have no toxic effect on the cells. Besides these desirable characteristics of PET/honey and PET/chitosan-honey mats, their well-known antibacterial properties sourced from the presence of chitosan and/or honey make them good alternatives as wound dressings. Further studies will be needed to compare wound-healing and antimicrobial capacity of these fibrous matrices. References [1] Zahedi P, Rezaeian I, Ranaei-Siadat SO, Jafari SH, Supaphol P. A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages. Polym. Adv. Technol. 2010;21:77–95. [2] Pulat M, Kahraman SA, Tan N, Gümüşderelioğlu M. Sequential antibiotic and growth factor releasing chitosan-PAAm semi-IPN hydrogel as a novel wound dressing. J. Biomat. Sci: Poly. Edn. 2013;24:807–819. [3] Pereira RF, Barrias CC, Granja PL, Bartolo PJ. Advanced biofabrication strategies for skin regeneration and repair. Nanomedicine. 2013;8:603–621. [4] Venugopal J, Ramakrishna S. Applications of polymer nanofibers in biomedicine and biotechnology. Appl. Biochem. Biotech. 2005;125:147–157. [5] Pereira RF, Bartolo PJ. Traditional therapies for skin wound healing. Adv. Wound Care. 2014. DOI: 10.1089/wound.2013.0506. [6] Gümüşderelioğlu M, Dalkıranoğlu S, Aydın RST, Çakmak S. A novel dermal substitute based on biofunctionalized electrospun PCL nanofibrous matrix. J. Biomed. Mater. Res. Part A. 2011;98A:461–472. [7] Jung K, Huh M, Meng W, Yuan J, Hyun S, Bae J, Hudson S, Kang I. Preparation and antibacterial activity of PET/chitosan nanofibrous mats using an electrospinning technique. J. Appl. Polym. Sci. 2007;105:2816–2823. [8] Ignatova M, Manolova N, Markova N, Rashkov I. Electrospun non-woven nanofibrous hybrid mats based on chitosan and PLA for wound-dressing applications. Macromol. Biosci. 2009;9:102–111. [9] Kim KW, Lee KH, Khil MS, Ho YS, Kim HY. The effect of molecular weight and linear velocity of drum surface on the properties of electrospun poly(ethylene terephthalate) nonwovens. Fibers Polym. 2004;5:122–127. [10] Hadjizadeh A, Ajji A, Bureau NM. Nano/micro electro-spun polyethylene terephthalate fibrous mat preparation and characterization. Mech. Behav. Biomed. 2011;4:340–351. [11] No HK, Park NY, Lee SH, Meyers SP. Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Int. J. Food Microbiol. 2002;74:65–72. [12] Raafat D, Bargen K, Haas A, Sahl HG. Insights into the mode of action of chitosan as an antibacterial compound. Appl. Environ. Microbiol. 2008;74:3764–3773. [13] Zhou Y, Yang D, Chen X, Xu Q, Lu F, Nie J. Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration. Biomacromolecules. 2008;9:349–354. [14] Sun K, Li ZH. Preparations, properties and applications of chitosan-based nanofibers fabricated by electrospinning. eXPRESS Polym. Lett. 2011;5:342–361. [15] Liao H, Qi R, Shen M, Cao X, Guo R, Zhang Y, Shi X. Improved cellular response on multi-walled carbon nanotube-incorporated electrospun polyvinyl alcohol/chitosan nanofibrous scaffolds. Colloids Surf., B. 2011;84:528–535. [16] Luo Y, Wang S, Shen M, Qi R, Fang Y, Guo R, Cai H, Cao X, Tomás H, Zhu M, Shi X. Carbon nanotube-incorporated multilayered cellulose acetate nanofibers for tissue engineering applications. Carbohydr. Polym. 2013;91:419–427. [17] Maleki H, Gharehaghaji AA, Dijkstra PJ. A novel honey-based nanofibrous scaffold for wound dressing application. J. Appl. Polym. Sci. 2013;127:4086–4092. [18] Dustmann JH. Antibacterial effect of honey. Apiacta. 1979;1:7–11.
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[19] Ozmen N, Alkın E. The antimicrobial features of honey and the effects on human health. Uludag Bee J. 2006;1:155–160. [20] Molan PCJ. The role of honey in the management of wounds. Wound Care. 1999;8: 415–418. [21] Veleirinho B, Rei FM, Lopes-DA-Silva JA. Solvent and concentration effects on the properties of electrospun poly(ethylene terephthalate) nanofiber mats. J. Polym. Sci., Part B: Polym. Phys. 2008;46:460–471. [22] Sill Travis J, von Recum Horst A. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials. 2008;29:1989–2006. [23] Deitzel JM, Kosik W, McKnight SH, Tan NCB, DeSimone JM, Crette S. Electrospinning of polymer nanofibers with specific surface chemistry. Polymer. 2002;43:1025–1029. [24] Megelski S, Stephens JS, Chase DB, Rabolt JF. Micro- and nanostructured surface morphology on electrospun polymer fibers. Macromolecules. 2002;35:8456–8466. [25] Min B, Lee SW, Lim JN, You Y, Lee TS, Kang PH, Park WH. Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers. Polymer. 2004;45:7137–7142. [26] Kriegel C, Kit KM, McClements DJ, Weiss J. Electrospinning of chitosan–poly(ethylene oxide) blend nanofibers in the presence of micellar surfactant solutions. Polymer. 2009;50:189–200. [27] Přıdal A, Vorlová L. Honey and its physical parameters. Czech J. Anim. Sci. 2002;47: 439–444. [28] Aziz AN, Majid RS, Yahya R, Arof KA. Conductivity, structure, and thermal properties of chitosan-based polymer electrolytes with nanofillers. Adv. Powder Technol. 2011;22: 1345–1348. [29] Tiwari A, Singh V. Synthesis and characterization of electrical conducting chitosan-graftpolyaniline. eXPRESS Polym. Lett. 2007;1:308–317. [30] Ma Z, Kotaki M, Yong T, He W, Ramakrishna S. Surface engineering of electrospun polyethylene terephthalate (PET) nanofibers towards development of a new material for blood vessel engineering. Biomaterials. 2005;26:2527–2536. [31] Xu H, Ma L, Shi H, Gao C, Han C. Chitosan–hyaluronic acid hybrid film as a novel wound dressing: in vitro and in vivo studies. Polym. Adv. Technol. 2007;18:869–875. [32] Tshukudu GM, van der Walt M, Wessels Q. Comparative in vitro study of honey-based and silver-based wound preparations on cell viability. Burns. 2010;36:1036–1041. [33] Barui A, Khare R, Dhara S, Banerjee P, Chatterjee J. Ex vivo bio-compatibility of honeyalginate fibrous matrix for HaCaT and 3T3 with prime molecular expressions. J. Mater. Sci. Mater. Med. 2011;1:1–9.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process a
b
c
Aysu Arslan , Murat Şimşek , Sevcan Dalkıranoğlu Aldemir , Nur d
Merve Kazaroğlu & Menemşe Gümüşderelioğlu
abd
a
Department of Chemical Engineering, Hacettepe University, Ankara 06800, Turkey b
Department of Nanotechnology and Nanomedicine, Hacettepe University, Ankara 06800, Turkey c
Department of Biology, Hacettepe University, Ankara 06800, Turkey d
Department of Bioengineering, Hacettepe University, Ankara 06800, Turkey Published online: 20 May 2014. To cite this article: Aysu Arslan, Murat Şimşek, Sevcan Dalkıranoğlu Aldemir, Nur Merve Kazaroğlu & Menemşe Gümüşderelioğlu (2014) Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process, Journal of Biomaterials Science, Polymer Edition, 25:10, 999-1012, DOI: 10.1080/09205063.2014.918455 To link to this article: http://dx.doi.org/10.1080/09205063.2014.918455
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 10, 999–1012, http://dx.doi.org/10.1080/09205063.2014.918455
Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process
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Aysu Arslana, Murat Şimşekb, Sevcan Dalkıranoğlu Aldemirc, Nur Merve Kazaroğlud and Menemşe Gümüşderelioğlua,b,d* a Department of Chemical Engineering, Hacettepe University, Ankara 06800, Turkey; Department of Nanotechnology and Nanomedicine, Hacettepe University, Ankara 06800, Turkey; cDepartment of Biology, Hacettepe University, Ankara 06800, Turkey; dDepartment of Bioengineering, Hacettepe University, Ankara 06800, Turkey b
(Received 23 December 2013; accepted 23 April 2014) In this study, fibrous mats were fabricated via electrospinning from solutions of polyethylene terephthalate (PET), PET/chitosan, and PET/honey at different concentrations. The effect of honey and chitosan on electrospinning process was investigated and compared. Fibers containing chitosan had a beaded or ribbon-like/ branched morphology, but this morphology improved in the presence of honey. The diameter of electrospun fibers decreased with an increased ratio of honey in PET solution. In addition, fiber deposition area in the collector increased by increasing the honey content. PET/chitosan and PET/honey fibrous mats reached an equilibrium water content in 15 min and their water uptake capacities, which are important for exudating wounds, were found in the range of 280–430% on dry basis. Cytotoxicity evaluation demonstrated that fibers exhibited no cytotoxic activity. This study discloses that PET fibrous mats especially electrospun in the presence of honey could be proposed as potential wound dressing materials owing to their improved processing abilities besides their suitable structural properties. Keywords: electrospinning; fibers; polyethylene terephthalate; chitosan; honey; wound dressing
1. Introduction Since the Egyptian times, a suitable material has been required to cover a wound for preventing any infection and thus for performing effective wound healing. Throughout history, honey pastes, plant fibers, and animal fats had been used as wound dressings.[1] Wound dressings are usually used to encourage the various stages of wound healing by creating better healing conditions.[2,3] An ideal wound dressing material is expected to have the following properties: (i) maintaining the most suitable environment at the wound/dressing interface, (ii) absorbing excess exudates, (iii) providing thermal insulation, (iv) providing mechanical and bacterial protection, (v) allowing gaseous and fluid exchanges, (vi) to be non-allergic, and non-scarring.[1,4] The wound-dressing materials produced by the electrospinning method have excellent properties when they were compared with the dressings obtained by conventional methods, including the preparation of polymeric films and foams.[2,5] Nanofibrous wound dressings having small holes and high effective surface area can promote *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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hemostasis phase.[1] Due to the high surface area to volume ratio of the nanofibers, they exhibit good water absorption capacity. Thus, if hydrophilic polymer fibers are employed, the fibrous dressings have a capacity to absorb wound exudates more efficiently than the conventional ones. Also, bacterial infection can be effectively inhibited due to the small pore size. Electrospun fibrous wound dressings can also meet the requirement of high gas permeation.[1] Various synthetic and natural polymers or their combinations have potential usage as electrospun nanofibrous wound dressings.[6] Among them, polycaprolactone (PCL), poly (L-lactic acid), polyvinyl alcohol, chitosan, and gelatin have been widely used.[7,8] Additionaly, in order to obtain electropun mats with desirable antibacterial characteristics two approaches have been used: (i) immobilization of drugs or antibacterial agents into the electrospun nanofibers and (ii) electrospinning of polymers which have intrinsic antibacterial and wound-healing properties, e.g. chitosan.[8] Polyethylene teraphthalate (PET) is a semicrystalline polyester that is widely used in the production of vascular grafts, films, fibers, containers, and engineering plastics. The properties of polyesters also make them potential candidates for producing fibers. Electrospun PET nanofibers were first obtained by Kim et al. [9] and nowadays, it has been used in electrospinning process to obtain nano/micro fibers especially for tissue engineering,[8,10] due to its advantageous mechanical properties, biocompatibility, and cost-effectiveness. Chitosan has excellent properties such as biodegradability, biocompatibility, and antibacterial and wound healing activity.[11–14] Recently, electrospun fibers based on chitosan have been widely investigated owing to their potential applications in various areas.[7,15,16] However, some difficulties may arise from the use of chitosan because its solutions could not be easily processed. Jung et al. [7] fabricated PET/chitosan electrospun fibrous matrices and they determined that these matrices had an antibacterial effect and biocompatibility. Honey is generally composed of glucose (30%), fructose (40%), sucrose (5%), water (20%), and many other substances, such as amino acids, vitamins, minerals, and enzymes.[17] It has been used in wound care, due to its medicinal properties. It is known that honey can provide a moist healing environment without the risk of bacterial infection.[18] Honey also has an antimicrobial property.[19,20] Maleki et al. [17] showed that fabrication of the electrospun honey-based fibrous matrices could be possible. They examined poly(vinyl alcohol) (PVA)/honey electrospun fibers and obtained uniform, bead-free fibers. In this study, PET, PET/chitosan, PET/honey, and PET/chitosan/honey solutions at different weight ratios were electrospun to obtain potential wound-dressing materials. The effect of chitosan and honey on fiber formation and morphology was compared. The resulting fibrous mats were well characterized in view of wettability, water-content, chemistry, and also cytotoxicity. 2. Materials and methods 2.1. Materials Chitosan (85% deacetylated, medium molecular weight) was supplied from SigmaAldrich. Trifluoroacetic acid (TFA) as a solvent for electrospinning was purchased from Merck. Commercial PET (MW: 49,500 g/mole) and honey from flowers in Meydancık country side of Artvin province in Turkey were used in the study.
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For cell-culture studies, 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) and penicillin-streptomycin solution were obtained from Sigma-Aldrich (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and trypsin/EDTA solution were purchased from Hyclone (US).
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2.2. Electrospinning of PET-based solutions Each electrospinning solution consisting of PET or PET/chitosan at certain weight ratios given in Table 1 was dissolved in TFA and stirred overnight at room temperature. For PET/honey solution (in TFA), complete dissolution time of PET increased up to 48 h with the increasing honey concentration from 10% to 40% (wt). Spinning solutions were transferred to a 2.5 mL syringe with a 21-gauge needle. Then, a high voltage (Gamma High Voltage Research, Ormond Beach, FL) was applied between the needle placed into syringe pump (NE 300, New Era Pump Systems) and stationary collector. Fibers were collected on an aluminium foil fixed onto the collector for a time period from 20 to 60 min to obtain approx. 140-μm thick fibrous matrices. 2.3. Characterization of fibrous mats Morphology of electrospun fiber mats was observed via a scanning electron microscope (SEM, Zeiss Evo 50, Germany). Fiber diameters were calculated using ImageJ software (NIH, Bethesda, MD) by taking the average of at least 100 measurements from SEM images. Attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) was performed to determine the presence of chitosan and honey in PET structure by using a Thermo Scientific Nicolet iS10 FTIR spectrophotometer (USA) bearing an ATR attachment with a diamond crystal. The water contact angles of electrospun fiber mats were measured by sessile drop method at room temperature (Kruss DSA 100, Germany) immediately after the deionized water droplet made contact with the fiber surface. Dynamic water uptake studies were carried out to determine equilibrium water contents of fiber mats. Experiments were done in excess phosphate buffer saline (PBS, pH: 7.4, 37 °C) for selected time intervals. The fibrous mats were soaked in PBS until
Table 1. Polymer concentrations and compositions used for the production of fibrous mats and their abbreviations. Final polymer concentration (%) 17
19
Compositions in 100 mL 15.3 mg PET + 1.7 mg chitosan 15.3 mg PET + 1.7 mg chitosan + 1.7 mg honey 17.0 mg PET + 1.7 mg honey 19.0 mg PET 17.1 mg PET + 1.9 mg chitosan 19.0 mg PET + 1.9 mg honey 19.0 mg PET + 3.8 mg honey 19.0 mg PET + 5.7 mg honey 19.0 mg PET + 7.6 mg honey
Abbreviations of fibrous mats 17(PETCs10) 17(PETCs10Hny10) 17(PETHny10) 19PET 19(PETCs10) 19(PETHny10) 19(PETHny20) 19(PETHny30) 19(PETHny40)
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they reached maximum water contents. Fibrous mats were removed from the swelling medium with certain time intervals, the excess liquid drops on the surface were wiped with a blotting paper and the weight of each mat was measured. The measurements were continued until the weight of the swollen mats reached a maximum value. Triplicate data were obtained for each measurement. The following equation (Equation 1) was used to calculate the mass swelling ratio based on dry weight.
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Q ¼ ½ðWs Wd Þ=Wd 100
(1)
where Ws is the weight of swollen sample and Wd is the weight of dried sample. The porosities of electrospun fiber mats were calculated using ImageJ software developed by the National Institutes of Health using ‘Auto Local Threshold’ plugin. The analysis was carried out via SEM images of electrospun mats having a similar thickness. 2.4. Cytotoxicity tests 2.4.1. Preparation of liquid extracts of mats The cytotoxicity of the electrospun fibrous mats was evaluated based on a procedure adapted from the ISO10993-5 standard test (MEM-extract test/MTT test system-72 h incubation) method. Experiments were carried out with L929 mouse fibroblasts obtained from HUKUK Cell Line Collection (No. 92123004, Foot and Mouth Disease Institute, Ankara, Turkey). PET, PET/chitosan, and PET/honey fibrous mats were compared in terms of cellular toxicity. Fiber mats were sterilized by ultraviolet (UV) light for 30 min. In this study 33 cm2 of sterilized PET, PET/chitosan and PET/honey fiber mats were extracted at 37 °C for 24 h in 35 mL culture medium. The medium consisted of DMEM with 10% (v/v) FBS, 1% penicillin-streptomycin, and 2.5% L-glutamine. After 24 h of incubation, fiber mats were removed from the medium and the extract was filtered using 0.22 μm filter (Orange Scientific). 2.4.2. Cell seeding and extract treatment Cell cultures were conducted in sterile 24-well tissue culture polystyrene (TCPS) dishes in stationary conditions. During the extract preparation, L929 cells were seeded into 24-well plates at an inoculation density of 5 × 104 or 6 × 104 cells/mL. After a 24 h incubation period the culture medium was replaced with various dilutions of the extract (25, 50, and 100%) to determine the cytotoxicity of fibrous mats by MTT assay and crystal violet staining. 2.4.3. Cell viability and growth kinetics of L929 cells Following 1st, 2nd and 3rd days of culture, the medium was removed and 600 μL prewarmed serum-free DMEM and 60 μL MTT solution (2.5 mg/mL dissolved in PBS) were added to each well which was incubated at 37 °C for 3 h. Then, the medium was removed from each well and 0.04 M HCl containing 400 μL isopropanol solution was added to dissolve formazan crystals. After pipetting the solution, violet colored 200 μL supernatant was transferred to 96-well plate and its optical density was measured spectrophotometrically at 570 nm with reference to 690 nm using a microplate reader (Asys UVM 340, Austria). Simultaneously, the morphology of the cells was observed
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by crystal violet staining. After 24 h incubation with the extract, the medium was removed from each well and washed two times with PBS. The cells were fixed in acetone-methanol solution (1:1) for 20 min. Then, the solution was removed and each well was treated with crystal violet solution (0.05% lyophilized crystal violet in 20% methanol) for 30 min. Each well was washed properly and examined under the inverted microscope (Olympus, USA).
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3. Results and discussion 3.1. Characterization of PET-based fibers Electrospinning conditions were optimized to obtain smooth and continuous fibers. For obtaining desirable fibrous mats, polymer concentration is a key parameter. For this reason, while the polymer (PET or PET/chitosan) concentration was varied from 15% (w/v) to 20% (w/v) to obtain smooth and continuous fibers, optimum process conditions were determined by changing the flow rate between 0.5 and 4.0 mL/h, applied voltage between 15 and 27 kV, and fiber collecting distance between 15 and 30 cm. The results revealed that discontinuous, coiled, or beaded fibers formed below 19% of polymer wt ratio. Above this concentration, electrospinning failed due to high viscosity, which is making the solution difficult to be ejected from a syringe. It was also observed that branched jet formation occured at a lower flow rate of 3 mL/h due to the formation of secondary jets. These jets could not reach the collector and thus it was difficult to obtain a stable Taylor cone which is essential for fiber production. The experiments showed that the following electrospinning parameters enabled best results in terms of fabrication of fibrous mats to be used: 19% (w/v) of polymer concentration, 3.5 mL/h of flow rate, 24 kV of applied voltage, and 35 cm of collecting distance. In this study, 17% (w/v) of PET/chitosan and PET/chitosan-honey solution were also used to observe the improvements in fiber morphology/production in the presence of honey. Table 1 shows polymer concentrations and compositions of solutions used for the production of fibrous mats with their abbreviations. Basic properties of fibrous mats are presented in Table 2. In order to investigate the effect of honey on the spinnability of PET/chitosan fibers, 10% (wt) of honey was added to the solution of 17(PETCs10) for preparing 17(PETCs10Hny10) matrix. Morphological differences between 17(PETCs10)
Table 2. Basic properties of fibrous mats (thickness: approx. 140 μm) prepared from different PET, chitosan, and honey compositions at the optimized electrospinning condition (voltage: 24 kV; flow rate: 3.5 mL/h; distance to collector: 30 cm). Composition of fibers 17(PETCs10) 17(PETCs10Hny10) 17(PETHny10) 19PET 19(PETCs10) 19(PETHny10) 19(PETHny20) 19(PETHny30) 19(PETHny40)
Fiber (nm)
Matrix porosity (%)
Equilibrium water content (%)
Water content angle (°)
565 + 245 557 + 217 678 + 145 682 + 111 1484 + 389 780 + 106 755 + 128 720 + 130 668 + 177
50.54 47.27 49.36 47.86 43.39 44.21 45.39 49.03 49.07
nd nd nd 430 + 20 426 + 21 418 + 27 nd nd 282 + 9
nd nd nd 135.0 + 1.5 0 134.8 + 1.3 nd nd 132.7 + 2.1
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Figure 1. SEM images of PET, PET/chitosan (PETCs) fibrous mats, and their honey-including forms (PETHny and PETCsHny, respectively) in different forms. (a) 17(PETCs10) (1KX), (b) 17 (PETCs10Hny10) (2KX), (c) 17(PETHny10) (2KX), (d) 19PET, (e) 19(PETCs10) (1KX), and (f) 19(PETHny40) (2KX). The photographs show higher magnifications.
and 17(PETCs10Hny10) fiber mats can be clearly seen from Figure 1(a) and (b). While the 17(PETCs10) fibers having a beaded morphology, the stability of the jet was enhanced in favor of the formation of bead-free fibers with the addition of honey. Since higher amount of solvent molecules in 17(PETCs10) than that of 19(PETCs10) caused an increase in the surface tension of polymer solution at this concentration (17% w/v), bead formation along with fiber structure appeared because of insufficent chain entanglement. However, no significant change in their fiber diameters was observed (Table 2). Figure 1(c) shows the SEM image of 17(PETHny10) fibers. Unlike 17(PETCs10) fibers, smooth 17(PETHny10) fibers were obtained in the presence of honey.
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Figure 1(d) shows that 19PET fibers have smooth and regular morphology with a diameter of 682 ± 111 nm. Veleirinho et al. [21] produced continuous nanofiber structures from 10% to 30% (w/v) PET solution by changing solvents (dichloromethane and TFA) and their volume ratios. They reported that fibers spinned from 30% w/v of PETTFA solution exhibited irregular morphology with branches or twigs. In this study, we used TFA as a solvent; however, in our optimized conditions, we did not observe similar defects related to PET fibers. In order to investigate the effect of chitosan on fiber morphology, 19% (w/v) of PET/chitosan solution (19(PETCs10)) was prepared. 19(PETCs10) fibers having 1484 ± 389 nm of diameter had partly ribbon-like and branched morphology when compared to 19PET fibers (Figure 1(e)). Also, it was seen that relatively very thin fibers arose from secondary jets separating main jet existed through the mat and they caused an increase in heterogenity of fiber diameter. Although polymer concentrations of 19PET and 19(PETCs10) were equal to each other, the fiber diameters increased in the presence of chitosan. This can be attributed to the polycationic structure of chitosan which has a significant effect on decreasing bending and stretching of the jet due to viscoelastic forces dominating the Columbic forces.[22–24] Similar results were reported in the relevant literature. Min et al. [25] stated that continuous fiber formation was inhibitied by repulsive forces between ionic groups within chitosan backbone under a high electric field. Ignatova et al. [8] reported that the electrospinning of poly[(L-lactide)-co-(D,Llactide)] (PLA) solution led to the formation of defect-free fibers, while the electrospinning of PLA/chitosan was causing broader distribution of fiber diameters than that of PLA fibers. In addition, in the case of PLA/chitosan systems splitting and branching of the fibers sometimes occurred. They explained that the elongation of the jet and the evaporation of the solvent could cause some shift in the balance between the surface tension and electrical forces and could result in jet splitting or branching of the fibers. In order to enhance the spinnability of chitosan and to obtain smooth fibers, some agents can be used as surfactants. Kriegel et al. [26] prepared PEO/chitosan fibers in the presence of surfactant molecule, sodium dodecyl sulphate, and reported that spinnability of jet was enhanced and smooth fibers were obtained. In order to examine the effect of honey on PET fibers, 19% (w/v) of PET/honey fibers were produced by increasing the honey ratio from 10 to 40% wt in the PET solution (Table 1). At a higher honey ratio than 40% wt, it was difficult to dissolve PET, completely. PET/honey fibers exhibited smooth and uniform fiber formation similar to PET fibers (Figure 1(f)). Maleki et al. [17] also produced uniform and smooth PVA/ honey nanofibers, independent of the honey content. In our study, fiber diameters slightly decreased with increasing honey content in spinning solution (Table 2). A decrease in fiber diameter can be explained with solution conductivity. If conductivity of a solution increases, more charges will be carried by the electrospinning jet. An increase in charge density results in a greater bending instability and stretching forces hence, fiber diameter decreases.[22] Since the conductivity of honey (90–130 × 10−5 S cm−1) is higher than that of chitosan (10−10 to 10−9 S cm−1),[27–29] a decrease in diameter of 19(PETHny10) fibers was observed. Fiber deposition area was also strongly affected by the honey ratio, inducing greater bending instability and stretching forces. 19(PETHny10) and 19(PETHny40) fibers had a deposition area of about 217 and 380 cm2, respectively, while the area was about 56 cm2 for 19(PETCs10) fibers. Therefore, collection time was increased from 20 to 60 min for obtaining fibrous mats having 140 ± 8 μm of thickness. Results showed that honey had a considerable effect on improving fiber production and fabrication during electrospinning process.
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Porosities of fiber mats are listed in Table 2. It can be seen that fiber porosities are almost the same, approx. 50%.
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3.2. ATR-FTIR analysis In ATR-FTIR spectrum of 19(PETCs10) matrix (Figure 2), O bridge stretching of the glucosamine residue and asymmetrical C–H bending of the CH2 group which belongs to chitosan were observed at 1042 cm−1 and 1371 cm−1, respectively. Figure 3 shows ATR-FTIR spectra of PET pellets, honey, and 19(PETHny40) mats. Since honey consists of mainly hydrated glucose and fructose, together with higher carbohydrates in smaller amounts, typical IR spectrum of honey is dominated by the features related to hydroxyl groups of carbohydrates, along with the bands due to skeletal vibrations of carbohydrates. The absorption of less abundant honey components is mainly overlapped. Compared to PET pellets, 19(PETHny40) fibers have additional characteristic peaks including C–H stretching at 2963 cm−1 (carboxylic acids and NH3+ of free amino acids), O–H stretching at 3420 cm−1 (water and carbohydrate), and other characteristic peaks (600–1500 cm−1) of carbohydrates, which indicates the presence of honey. 3.3. Wettability and water contents of fibrous mats Water contact angles of fiber mats were measured immediately after the water droplet made contact with the fiber surface. Table 2 gives the water contact angles of 19PET, 19(PETCs10), 19(PETHny10), and 19(PETHny40) fiber mats. While the contact angles of PET and PET/honey fibers are almost same, the contact angle of chitosan-blended mat, i.e. 19(PETCs10) mat, was found to be zero because of strong water affinity of chitosan. Fluid affinity is a very important parameter in terms of fluid absorption of a
Figure 2.
ATR-FTIR spectra of PET pellets, chitosan flakes, and 19(PETCs10) fibrous mats.
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ATR-FTIR spectra of PET pellets, honey, and 19(PETHny40) fibrous mats.
wound-dressing material especially for exudates wounds.[1] In a study,[30] although PET nanofibers had about 128° of the water contact angle, they became a highly wettable material (0°) after surface modification either by poly(methacrylic acid) grafting or by gelatin grafting. Equilibrium water contents of fibrous mats are given in Table 2. Although PET and PET/honey fibers exhibited hydrophobic behavior at the time of water contact with fiber surface, they absorbed water in a swelling medium. This behavior can be attributed to the porous structure of the fibrous mats allowing water access. All mats except 19PET swelled rapidly in 15 min. 19(PETCs10) and 19(PETHny40) fibers reached a maximum water uptake capacity at that period, whereas it took 180 and 90 min for 19PET and 19(PETHny10) fibers, respectively. Although conventional film dressings only show water absorption of 2.3%,[1] our PET-based fiber mats exhibited the absorption up to 280%. Xu et al. [31] prepared wound-dressing materials using chitosan/hyaluronic acid and examined their water uptake capacity. They found that the dressing materials had water content in the range of 200–450% in dry basis.
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3.4. Cytotoxicity evaluation In order to find out if the fibrous mats release toxic products or not, cytotoxicity test was carried out. The cytotoxicities of PET, PET/chitosan and PET/honey fibrous mats were determined by testing the viabilities of L929 cells through the colorimetric estimation using MTT assay. In this test, after exposure to the various dilutions of extract (i.e. 25, 50, and 100%), proliferation of L929 cells was measured for 3 days. As a control, no extract-added cell cultures were used. Absorbance values are evaluated as the measure of cellular proliferation. The extracts of the mats showed no inhibition of cell metabolism compared to the control group (Figure 4). Mitochondrial activities of L929 cells cultured with mat extracts have no statistically significant difference from control groups although the average absorbance values were less than that of the control group. In addition, cell death and morphological damage were not observed throughout the incubation period. Figures 5 and 6 present the crystal violet images of L929 cells at the first and second days of culture at different concentrations of PET, PET/chitosan, and PET/honey extract media. The number of cells has increased with time as expected. However, cells that are cultured with extracts of PET/honey (10% and 40% wt of honey) mats are more spindle-shaped and less confluent than other groups at the first day of culture period. As seen from Figure 5, these cells have elongated morphology when compared with cells incubated in PET and 19(PETCs10) extract groups. On the second day of
Figure 4. Mitochondrial activities of L929 fibroblasts proliferated in the presence of 0, 25, 50, and 100% extracts of the fibrous mats. (a) 19PET, (b) 19(PETCs10), (c) 19(PETHny10), (d) 19(PETHny40). Statistically significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001).
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Figure 5. First day crystal violet images of L929 fibroblasts proliferated in the presence of 0, 25, 50, and 100% extracts of the fibrous mats. (a–d) 19PET, (e–h) 19(PETCs10), (i−l) 19(PETHny10), and (m–p) 19(PETHny40) (magnifications are ×40).
culture (Figure 6), the group treated with extract of 19(PETHny10) started to gain normal morphology similar to PET, 19(PETCs10) extract groups, and control groups. On the other hand, the cells treated with the extract of 19(PETHny40) mats continued to protect their spindle-shaped morphology. However, at the end of the third day these cells also gained normal morphology (photographs were not given). These type of morphological changes arising from honey had been previously reported on the human keratinocyte cells that showed distinct cytoplasmic projections.[32] Additionally, Barui et al. [33] successfully investigated the role of honey on the expression of Type I and Type III collagens for 3T3 fibroblast cells and E-Cadherin for HaCaT cells during the cell–cell and cell–material interactions on the honey-alginate fibrous mat. They prepared fibrous honey-alginate mat via wet-spinning technique through physical entrapment of honey into alginate fibers. In the presence of honey, higher cellular viability and maturation have been observed on the alginate structure. Immuno-cytochemical assays demonstrated higher expression of collagen I and collagen III on the honey-alginate mat. Honey-including alginate scaffold induced to gain of functional dimensions
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Figure 6. Second day crystal violet images of L929 fibroblasts proliferated in the presence of 0, 25, 50, and 100% extracts of the fibrous mats. (a–d) 19PET, (e–h) 19(PETCs10), (i–l) 19(PETHny10), and (m–p) 19(PETHny40) (magnifications are ×40).
on 3T3 fibroblasts, as dependent on collagen synthesis. On the other hand, enhanced membranous expressions of E-cadherin in HaCaT cells on the honey-based materials demonstrated better impact in establishing cell–cell adhesion in the state of confluence. When considered together with the results of cytotoxicity assay and morphological imaging, electrospun PET/honey mats have the potential to be used as a wound-closure material. 4. Conclusions In the presented study, as potential wound-dressing candidates PET, PET/chitosan, and PET/honey fibers were electrospun and their physical structures were examined. Smooth and uniform PET/honey fibers were obtained up to 40 wt% honey. In the case of PET/chitosan, while the fibers had a beaded morphology at a lower polymer concentration (17% w/v), fiber diameter increased and ribbon-like/branched fibers were observed at a higher polymer concentration (19% w/v). The jet stability was improved
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and processing limitations related to the spinning of chitosan were eliminated with the addition of honey to the spinning solution. All fiber mats showed good water-absorbing capacity when compared to typical wound dressings. MTT results demonstrated that electrospun PET, PET/chitosan, and PET/honey matrices have no toxic effect on the cells. Besides these desirable characteristics of PET/honey and PET/chitosan-honey mats, their well-known antibacterial properties sourced from the presence of chitosan and/or honey make them good alternatives as wound dressings. Further studies will be needed to compare wound-healing and antimicrobial capacity of these fibrous matrices. References [1] Zahedi P, Rezaeian I, Ranaei-Siadat SO, Jafari SH, Supaphol P. A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages. Polym. Adv. Technol. 2010;21:77–95. [2] Pulat M, Kahraman SA, Tan N, Gümüşderelioğlu M. Sequential antibiotic and growth factor releasing chitosan-PAAm semi-IPN hydrogel as a novel wound dressing. J. Biomat. Sci: Poly. Edn. 2013;24:807–819. [3] Pereira RF, Barrias CC, Granja PL, Bartolo PJ. Advanced biofabrication strategies for skin regeneration and repair. Nanomedicine. 2013;8:603–621. [4] Venugopal J, Ramakrishna S. Applications of polymer nanofibers in biomedicine and biotechnology. Appl. Biochem. Biotech. 2005;125:147–157. [5] Pereira RF, Bartolo PJ. Traditional therapies for skin wound healing. Adv. Wound Care. 2014. DOI: 10.1089/wound.2013.0506. [6] Gümüşderelioğlu M, Dalkıranoğlu S, Aydın RST, Çakmak S. A novel dermal substitute based on biofunctionalized electrospun PCL nanofibrous matrix. J. Biomed. Mater. Res. Part A. 2011;98A:461–472. [7] Jung K, Huh M, Meng W, Yuan J, Hyun S, Bae J, Hudson S, Kang I. Preparation and antibacterial activity of PET/chitosan nanofibrous mats using an electrospinning technique. J. Appl. Polym. Sci. 2007;105:2816–2823. [8] Ignatova M, Manolova N, Markova N, Rashkov I. Electrospun non-woven nanofibrous hybrid mats based on chitosan and PLA for wound-dressing applications. Macromol. Biosci. 2009;9:102–111. [9] Kim KW, Lee KH, Khil MS, Ho YS, Kim HY. The effect of molecular weight and linear velocity of drum surface on the properties of electrospun poly(ethylene terephthalate) nonwovens. Fibers Polym. 2004;5:122–127. [10] Hadjizadeh A, Ajji A, Bureau NM. Nano/micro electro-spun polyethylene terephthalate fibrous mat preparation and characterization. Mech. Behav. Biomed. 2011;4:340–351. [11] No HK, Park NY, Lee SH, Meyers SP. Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Int. J. Food Microbiol. 2002;74:65–72. [12] Raafat D, Bargen K, Haas A, Sahl HG. Insights into the mode of action of chitosan as an antibacterial compound. Appl. Environ. Microbiol. 2008;74:3764–3773. [13] Zhou Y, Yang D, Chen X, Xu Q, Lu F, Nie J. Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration. Biomacromolecules. 2008;9:349–354. [14] Sun K, Li ZH. Preparations, properties and applications of chitosan-based nanofibers fabricated by electrospinning. eXPRESS Polym. Lett. 2011;5:342–361. [15] Liao H, Qi R, Shen M, Cao X, Guo R, Zhang Y, Shi X. Improved cellular response on multi-walled carbon nanotube-incorporated electrospun polyvinyl alcohol/chitosan nanofibrous scaffolds. Colloids Surf., B. 2011;84:528–535. [16] Luo Y, Wang S, Shen M, Qi R, Fang Y, Guo R, Cai H, Cao X, Tomás H, Zhu M, Shi X. Carbon nanotube-incorporated multilayered cellulose acetate nanofibers for tissue engineering applications. Carbohydr. Polym. 2013;91:419–427. [17] Maleki H, Gharehaghaji AA, Dijkstra PJ. A novel honey-based nanofibrous scaffold for wound dressing application. J. Appl. Polym. Sci. 2013;127:4086–4092. [18] Dustmann JH. Antibacterial effect of honey. Apiacta. 1979;1:7–11.
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