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Electrospun zwitterionic nanofibers with in situ decelerated epithelialization property for non-adherent and easy removable wound dressing application
Chemical Engineering Journal 287 (2016) 640–648
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Electrospun zwitterionic nanofibers with in situ decelerated epithelialization property for non-adherent and easy removable wound dressing application Afeesh Rajan Unnithan a,b,1, Amin Ghavami Nejad a,1, Arathyram Ramachandra Kurup Sasikala a, Reju George Thomas c, Yong Yeon Jeong c, Priya Murugesan d, Saeed Nasseri d, Dongmei Wu d, Chan Hee Park a,b,⇑, Cheol Sang Kim a,b,⇑ a
Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju, Republic of Korea Division of Mechanical Design Engineering, Chonbuk National University, Jeonju, Republic of Korea c Department of Radiology, Chonnam National University, Hwasun Hospital, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea d Department of BIN Fusion Technology, Chonbuk National University, Jeonju, Republic of Korea b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Non cell adherent wound dressing
membranes. Easy removable, no-pain wound
dressing bandages. Blood inert wound dressing
membrane, suitable for large areas of chronic wounds. Resist microbial biofilm formation and hence provides minimum chance of infection. Minimum cosmetic scar due to less cell adsorption on wound dressing membrane.
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 6 November 2015 Accepted 25 November 2015 Available online 28 November 2015 Keywords: Zwitterion Carboxybetaine Electrospinning Wound dressing Nanomembrane Electrospinning
a b s t r a c t Wound care management is a serious issue among the medical practitioners due to its varying complexity and various materials were tested for fast relief and easy removal. In this regard zwitterionic polymer based wound dressing membranes are the key point of attraction. Here we prepared a novel zwitterionic poly (carboxybetaine-co-methyl methacrylate) (CBMA) copolymer based nanomembranes using the electrospinning technique for the wound dressing application. The study takes advantage of the outstanding chemical properties of zwitterionic CBMA and the morphological efficiency of nanomembranes. The cell attachment studies proved the cell inert nature of thus prepared membranes. Such non cell adherent wound dressing membranes can be applied as the easy removable, no-pain wound dressing bandages. Our results clearly showed that the excellent blood-inert nature can be achieved by the CBMA nanofiber membranes. Therefore, there will be less chance of attaching blood clot with the wound dressing membrane and is extremely significant for the care of patients with large areas of chronic wounds. Additionally the in vivo results showed the formation of new tissues within two weeks, evidence of a complete wound healing material. So our CBMA membrane can be successfully used as a perfect wound dressing material with minimum cosmetic scar. Ó 2015 Elsevier B.V. All rights reserved.
⇑ Corresponding authors at: Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju, Republic of Korea. 1
E-mail addresses: [email protected] (A.R. Unnithan), [email protected] (C.H. Park), [email protected] (C.S. Kim). These authors contributed equally.
http://dx.doi.org/10.1016/j.cej.2015.11.086 1385-8947/Ó 2015 Elsevier B.V. All rights reserved.
A.R. Unnithan et al. / Chemical Engineering Journal 287 (2016) 640–648
1. Introduction The skin is considered to be the largest organ of the body, and is serves many different functions. Therefore, wound care management is a serious healthcare issue. Wound dressings are primarily responsible for managing the most types of open wounds [1]. The immediate care of skin wounds is important to prevent microbial infections as well as trans-epidermal water loss that may cause acceleration in the wound regeneration [2]. Therefore, the restoration of the skin barrier is of crucial importance for the treatment of injuries [3]. Wound dressing materials made via electrospinning technique have been increasingly studied due to their superior properties, and the electrospun nanofibers have been appropriate for use as perfect wound dressing materials as a result of their useful properties, including flexible pore-size distribution, high porosity to provide oxygen permeability, and high surface-to-volume ratio [4–6]. A variety of materials have been electrospun to control the microenvironment of the wound surface as well as to stimulate the healing process, and currently nonadherent, wound dressing materials have been preferred in the sense that they can be easily removed without trauma, possesses antimicrobial properties and promote wound healing with minimal scarring [7–10]. Natural biopolymers, such as chitosan [11,12], alginate [13], cellulose acetate [14] and, hyaluronic acid [15] have been extensively studied for their potential role as materials for wound dressings. The use of such biopolymers is sometimes limited due to their dissimilarities from batch to batch of raw materials and the possibility of the transmission of an infection due to their bioactive nature. Many synthetic polymers, such as polyurethane [10,14], poly (e-caprolactone), and poly (L-lactide) [16], have also been used as wound dressings. The mechanical properties, desirable cytocompatibility and low cost of the synthetic polymers affirm that these are perfect materials for use in wound dressing applications [17]. However, most polymeric materials lack resistance to the nonspecific adsorption of proteins. This can result in nonspecific cell attachment and in bacterial adhesion, which in turn would cause bacterial infections and pain upon removing the wound dressing material as a result of the cell growth over the material [18]. Recent studies have shown that a zwitterionic copolymer composed of carboxybetaine methcrylate monomer exhibits excellent blood compatibility, including the suppression of platelet adhesion and complement activation on the surface of the polymer [19]. They are also known to be resistant to cell adhesion, both in vitro and in vivo [20]. The zwitterionic carboxybetaine polymers have also been reported to possess antibacterial properties and resistance to the formation of bacterial films [21,22]. Their desirable properties, such as a strong resistance to protein adsorption, cell attachment, and resistance to bacterial growth, have made carboxybetaine methcrylate polymers promising materials for use as nonadherent wound dressing materials [23]. Such membranes will not damage healing tissue and can be easily applied and removed, causing no pain [18]. Furthermore, electrospinning is an easy, efficient method that can be used to prepare such nanofibrous membranes. The electrospun nanofibers have been found to be very effective when used as wound dressing materials mainly due to their architectural superiority [24]. We synthesized poly (carboxybetaine-co-methyl methacrylate) copolymer (denoted as CBMA throughout this paper). The application of the methylmethacrylate based material as a wound dressing has already been extensively reported [25]. In this work, a fibrous membrane was electrospun from as-prepared CBMA polymer for use as a wound dressing. The main objective of this study is to develop an electrospun nanofibrous membrane from CBMA in order to study its properties when intended for use as active non-
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adherent wound dressings. The cell and platelet adhesion behavior and the in vivo wound healing efficiency of the zwitterionic CBMA nanofibrous membrane were thus studied. 2. Materials and methods 2.1. Synthesis and characterization of the carboxybetaine monomer [CBAA-5-ester: (6-carboxypentyl)-3-acrylamidopropyl dimethylammonium bromide, ethyl ester] The carboxybetaine monomer was prepared and characterized following methods that were previously reported [26]. N-(3Dimethylaminopropyl) acrylamide (50 mmol), ethyl 6-bromohexanoate (55 mmol), and acetonitrile (25 mL) were added into a 100-mL round-bottom flask. The mixture was stirred under a nitrogen atmosphere for five days at 45 °C. The solvent was removed on a rotary evaporator, and the product was then precipitated and washed with anhydrous ether. In the next step, 100 mg/mL of monomers were dissolved into aqueous solutions with 1 M NaOH concentrations. After 24 h of hydrolysis, the solution was neutralized with a dilute HCl solution and water was then removed by vacuum. 1H-NMR (400 MHz, D2O): 2.00 (m, 4H, CACH2AC), 2.47 (t, 2H, CH2AC@O), 3.1 (s, 6H, N+(CH3) 2, 3.3–3.4 (6H, CHAN and CH2AN + ACH2), 5.75 (m, 1H, CH@CACON-trans), 6.19 (m, 1H, CH@CACON-cis), 6.26 (m, 1H,@CHACONA). 2.2. Synthesis of poly (carboxybetaine -co-methyl methacrylate) copolymer abbreviated as CBMA An appropriate amount of two monomers, MMA (47 mmol) and carboxybetaine monomer (4.7 mmol), were added to a 50-mL round bottomed flask that contained 30 mL of dimethylformamide. The solution was bubbled with nitrogen for 10 min, azobisisobutyronitrile was then added to the flask, and the solution was heated up to 70 °C and was stirred overnight. Next, the solution was added drop-wise to 400 mL of diethyl ether while stirring to precipitate the synthesized copolymer. The purified copolymer was dried overnight in a vacuum oven at room temperature. The final products present a white to light yellow color and had a yield of 68%. The molar ratio of carboxybetaine to MMA in the obtained copolymer was estimated to be 9.8% by 1H-NMR analysis (Fig. 1 supporting information) [27,28] by taking the integral of a peak at 3.4 ppm belongs to the six protons present in the two methyl groups attached to the quaternary amine ((CH3)2N+) of carboxybetaine pendant group, and a peak at 3.7 ppm belongs to the three protons present in the terminal methyl group of the MMA pendant group. It should be mention that deuterated chloroform was used to dissolve copolymer and which results in the sharp peak at 7.24 ppm. The materials were also tested chromatographically, but due to the poor solubility of the materials, it was not possible to provide exact values, even with the addition of salts such as LiCl [29]. The carbobetaine repeat unit of the copolymer is hydrophilic, and makes the polymer insoluble in the relatively hydrophobic solvents [29]. However, since the polymer solution is spinable it can be said the molar masses are high enough for the electrospinning process. 2.3. Fabrication of the CBMA nanofibers Copolymer solutions were prepared by dispersing the polymer at concentration of 20 wt% in dimethyl formamide (DMF). The obtained solutions were placed in a plastic syringe tube and were fed through a metal capillary (nozzle) with a diameter di = 0.21 mm (21 G) attached to a 1-D robot system that moves laterally and is controlled by the LabVIEW 9.0 software program
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(National Instruments). A controllable syringe pump was used to maintain the feeding rate at 0.5 ml/h. Electrospinning was carried out at a voltage of 18 kV and a working distance of 15 cm at room temperature. 2.4. Rheological characteristics The mechanical properties of the polymeric solution were tested using a rheometer (Malvern KinexusPro) with a 20 mm/1° or 2° cone/plate geometry. 2.5. Characterization of the nanofibers Field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan) was used to observe the morphology of the electrospun mats. 2.6. Platelet activation study Blood clotting studies were conducted according to methods previously reported in the literature [30]. Blood was drawn from a voluntary human ulnar vein using a BD Discardit II sterile syringe and was mixed with anticoagulant agent acid citrate dextrose at a ratio of 9:1. Triplicate samples were used for this study, and a pristine PMMA mat was used as the negative control. Blood was added to each of the composite nanofiber mats and was placed in a 25-mL plastic Petri dish, followed by the addition of 10 lL of 0.2 M CaCl2 solutions to initiate blood clotting. These mats were then incubated at 37 °C for 10 min. 15 mL of distilled water were then added drop-wise without disturbing the clot. Subsequently, 10 mL of solution were taken from the dishes and were centrifuged at 1000 rpm for 1 min. The supernatant for each sample was collected and was kept at 37 °C for 1 h. Two hundred microliters (200 lL) of this solution were transferred to a 96-well plate. The optical density at 530 nm was measured using a plate reader (Dynex Technologies USA). For the platelet activation studies, blood was mixed with anticoagulant and was centrifuged at 2500 rpm for 10 min. The supernatant rich in platelet plasma was collected and was added directly to the nanofibers. The samples were fixed with glutaraldehyde and were then washed with PBS. Later these samples were dehydrated using a gradient alcohol treatment and were then viewed under SEM. 2.7. Antibacterial activity measurement with a disc diffusion test All of the bacteria used in this study were purchased from a Korean type culture collection. Escherichia coli (KACC 14818) and Staphylococcus aureus (KACC 10768) were obtained, and all cultures were maintained in brain heart infusion agar (BHIA) plates and were stored at 4 °C. An inhibitory test of CBMA nanofibers was conducted by using the disc diffusion method [31]. The CBMA nanofibrous mats and the pristine PMMA nanofibers were cut into small circular discs with a diameter of around 5 mm each and were denoted as A and B, respectively. These discs were then placed on the surfaces of the petri dishes. The inhibition zones were estimated after 0–240 min. CBMA- and PMMA-only discs were placed as control discs on the plates seeded with the test organisms. The antibacterial activity plates were then incubated at 37 °C, and the diameters of the inhibition zones were measured using a transparent ruler. 2.8. Biocompatibility study The CBMA nanofibers were seeded with 5 104 cells of the NIH3T3 cell line (mouse embryonic fibroblast cells) into 24-well plates and were cultured in DMEM medium in a 5% CO2 incubator
with a humidified environment at 37 °C. The measurements for the nanofiber were taken in triplicate along with the control samples that had cells seeded onto a coverslip (SPL Lifesciences, Korea) only. After 2nd, 4th and 6th day, 50 ll of MTS reagent (Promega, USA) were added to each of the wells and were incubated for 4 h. A microplate reader was used to measure the absorbance at 490 nm. For the cell attachment study, the scaffolds were rinsed twice with PBS and were subsequently fixed in 4% paraformaldehyde after the 2nd and 4th days of incubation. Next, the samples were rinsed with distilled water and were then dehydrated with a graded concentration of ethanol, i.e., 20%, 30%, 50%, 70% and 100% ethanol for 10 min each. Finally, the samples were kept in a vacuum oven and were then sputter-coated with gold to observe the cell morphology by using SEM. After 48 h of incubation, the cells were treated with cytoskeletal phalloidin staining with 200 ll of phalloidin stain (Molecular probes, USA) and DAPI dissolved in PBS for 30 min at room temperature. The coverslip containing the cells was viewed directly under a fluorescent microscope. 2.9. In vivo wound dressing study The in vivo animal study was approved by the Institutional Animal Ethical Committee of Chonbuk National University, Jeonju, South Korea (IACUC certification number CBNU 2014-00062). Wistar rats weighing 200–250 g and aged 4–6 weeks were used in this study. The rats were divided into two groups, with each group containing three rats (n = 3). The rats were allowed to take normal rat feed and water without restriction. On the day of the wounding, the rats were anaesthetized with an intraperitoneal injection of 80 mg/kg ketamine and 8 mg/kg xylazine. The dorsal area of the rats was depilated, and the operative area of the skin was cleaned with alcohol. A partial thickness skin wound with a size of 1.5 cm2 was prepared by excising the dorsum of the rat using surgical scissors and forceps. The prepared wounds were then covered with the CBMA nanofibers, and the rats with the bare wound were covered using cotton gauze. After the dressing materials were applied, the rats were individually housed in cages under normal room temperature. The dressing materials were changed at weeks 1, 2, and 3. During the change in the dressings the hair of the animals was cropped, and photographs were taken. The wound area was then measured using a transparent polyethylene sheet. The sheet was kept on top of the wound, and the area was marked using a marker pen. After week 3, the skin wound tissue of the rats was excised, fixed with 10% formalin, and stained with a hematoxylin eosin (H&E) reagent to conduct histological observations. 3. Results and discussion 3.1. Physical characterizations of composite nanofibers The FESEM images of electrospun CBMA nanofibers are shown in Fig. 1. These randomly oriented nanofibers exhibited a beadfree, smooth surface with almost uniform diameters along their lengths. The diameters of these composite nanofibers were determined to be in the range from 800 to 900 nm. The composite electrospun nanofiber mats produced in this study were desirably smooth and flexible and were successfully used in the desired studies. The porosity measurements using mercury intrusion method also confirmed the porous nature of the CBMA nanofiber mat (Fig. 3 supporting information). Thus, the electrospun CBMA nanofibrous scaffolds possess a perfect porous morphological appearance that is crucial for gaseous and fluid exchange in a wound environment. The water contact angle measurement showed the hydrophobic nature of the CBMA nanofiber mat. The
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Fig. 1. FESEM images of electrospun (A) and (B) CBMA nanofiber mat at lower and higher magnifications respectively.
PMMA nanofiber mat showed a water contact angle of 126.32° ± 2.5°, whereas CBMA mat showed 123.32° ± 1.8° as contact angle. A very small decrease in contact angle was observed in CBMA mat compared to PMMA nanofiber mat, even though both maintained the hydrophobic nature (Fig. 4 supporting information). 3.2. Cell attachment and biocompatibility study A critical property of wound dressing materials is their ability to resist the adhesion of cells instituting epidermis. The adhesion of fibroblasts to the wound dressing material delays healing, which results in an associated supplementary trauma. To observe the cell adhesion and repulsion, the morphological appearances of the cells on the composite nanofiber mats was obtained after 2 and 4 days of culture. Cell attachment studies were conducted using FESEM images (Fig. 2), and these revealed that the cells were attached to the electrospun PMMA nanofibrous scaffolds. In the case of the electrospun PMMA nanofibrous scaffolds, the cells began to spread on the nanofibers by the 4th day. The electrospun PMMA nanofibrous scaffolds served as a good environment for cell attachment and proliferation since they mimic the native extracellular matrix (ECM) morphology and have high area-to-volume ratio and porosity. On the other hand, the electrospun CBMA composite nanofibrous scaffolds showed a significantly low affinity for cell attachment. After 4 days of culture, the cells become rounded and were not able to spread on the surface of the electrospun zwitterionic CBMA nanofibrous scaffold (Fig. 2D). The MTT results also confirmed a lack of cell adhesion to the CBMA nanofibrous scaffold (Fig. 2E). The CBMA nanofibrous surfaces were devoid of any cell attachment proteins as a result of the zwitterionic content, and hence, the cells could not spread onto the CBMA composite nanofibrous scaffolds [32,33]. The improved resistance to cell adhesion was further confirmed via DAPI staining and confocal imaging. The nuclear stain DAPI showed a reduced cell growth in the CBMA nanofibrous scaffold relative to that of pure PMMA nanofibers, as shown in Fig. 3. Phalloidin staining was carried out to understand the response of the cells to the zwitterionic nanofibrous scaffold after 2 days of culturing, and the F-actin stains showed a cytoplasmic filamentous distribution under a confocal microscope. The cell density of the cells that proliferated in the control sample was higher than that in the CBMA nanofiber scaffold. We found that the cell density and morphology were better for the control sample than for the electrospun zwitterionic CBMA nanofibrous scaffolds (Fig. 3A and B). The cells attached to the control PMMA scaffolds and showed sufficient cell-to-cell communication. However, the cells that attached on CBMA nanofibrous scaffolds showed limited
cell–cell communication with a limited distribution of cells. As a result, the cell density was lower on the zwitterionic nanofibrous scaffold than that of the cells that proliferated on the control PMMA scaffold. Zwitterionic membranes are well known to be resistant to protein adsorption and, hence, repel cell adhesion [18,34]. The inert cell property of the nanofibrous mats can be an advantage for wound care management in many ways. Such non cell-adherent wound dressing membranes can be applied as easily removable, painless bandages [35]. These membranes will not cause any pain upon frequent removal, moreover the healing tissue will not be damaged [35,36]. Since the newly formed skin layer is not disturbed, a minimal scar can be expected when such wound dressings are used. 3.3. Platelet activation study The blood inert nature of the CBMA nanofiber mats was studied by conducting a platelet activation analysis. The SEM images showed that the PMMA nanofiber mats allowed the platelets to settle on the nanofiber surface, as seen in Fig. 4A. The SEM images showed that the platelets were spread all over the PMMA nanofibrous mat surface. The SEM results showed the full-scale platelet adhesion and activation at the blood contact site. It is clear that very few platelet attachments from whole blood were observed on the surface of the CBMA nanofiber mats when compared to the full-scale attachment and aggregation of platelets on the control nanofiber mats (Fig. 4B). The optical density measurement revealed that the CBMA nanofiber mats exhibited limited blood clotting ability when compared with pristine PMMA mats, as shown in Fig. 4C. Zwitterionic polymers were already known to have a blood inert nature [37,38], and these results clearly indicate that an excellent blood-inert nature can be achieved by CBMA nanofiber mats, which are shown in Fig. 4B to resist platelet adhesion. The electrostatic attraction between the charges on the pendant groups may contribute to the protein resistance of the CBMA nanofiber membranes. Preferably, the wound dressings should not activate blood defense systems or blood cells, and furthermore, if the surface of the wound dressing membrane interacts with any of the blood components, the wound surface will be at risk once the dressing is removed. Therefore, the probability that a blood clot attaches to the wound dressing membrane is reduced, and hence, non-adherent wound dressings are extremely important for the care of patients with large-area chronic wounds. 3.4. Antibacterial effects The prevention of a microbial infection is a key factor for any type of wound dressing material [39]. The CBMA nanofiber
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Fig. 2. FESEM images showing the cell attachment and morphology on PMMA nanofiber mats and CBMA nanofiber mats after day 2 (A and B) and day 4 (C and D) respectively, (E) MTT cell growth measurement assay. The viability of control cells was set at 100%, and viability relative to the control was expressed.
membranes exhibited a clear zone of inhibition with both Grampositive and Gram-negative bacterial strains. As shown in Fig. 5, the Gram-positive bacteria S. aureus presented a zone of inhibition with a mean diameter of around 15 mm, and the Gram-negative E. coli had an inhibition zone of nearly 18 mm. No bactericidal activity was detected for the pristine PMMA nanofibrous mats. The CBMA nanofiber membranes exhibited excellent bactericidal activity against a wide range of bacteria; therefore exogenous infections were effectively avoided. We observed a small zone of inhibition which might be due to the small molecular quaternary ammonium compounds (QMCs) with carboxybetaine esters. These QMCs could bind to the outer membrane and cytoplasmic membrane of bacteria and permeate into bacterial membranes [26]. The sanitization of exogenous organisms is a critical factor for an ideal wound dressing material. The physicochemical properties of the wound dressing material surface determine the adhesion of the bacteria to the materials. The zwitterionic content is already known to cause microbial resistance and so contributes to the microbial resistance in the CBMA nanofiber membranes. Thus, the CBMA nanofiber membranes that were prepared could be sued to combat biofilm formation, which can potentially affect most wound dressing materials [40]. It is crucial to do so in order to stop or reduce a bacterial infection in its initial stages, which can be life threatening. Therefore, the main advantage of using a CBMA
nanofiber membrane dressing is that it will not cause any fouling in the dressing due to the formation of the microbial biofilm. Hence, it provides a minimum chance of introducing new bacteria with repeated exposure of the wound site to the outside and also provides more comfort to the patients [41]. 3.5. In vivo wound dressing study The main purpose of developing the CBMA nanofiber membranes was to evaluate the skin tissue reconstruction in the wounds. An in vivo study was conducted with Wistar rats, and the wound healing ability of the prepared CBMA nanofibrous composite mat was proven. Fig. 6 shows photographs of the in vivo wound healing study. The CBMA composite mats achieved excellent healing in the first and second weeks relative to the bare wound. The control seemed to delay the wound recovery when compared to the CBMA mat dressings. The extent of the wound closure was evaluated and observed after 3 weeks, and the wounds that were treated with the CBMA mat significantly improved the closure at 92% when compared to bare wounds that showed a wound closure of 70% (Fig. 6). After 3 weeks of contact with the CBMA composite mat, the thickness of the granulation layer was similar to that of unwounded skin, indicating optimal healing. Besides that the qualitative histomorphology in routinely-stained
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Fig. 3. Fluorescent microscopic images of phalloidin staining of cells on (A) control nanofibers, (B) CBMA nanofibers respectively.
Fig. 4. SEM images of platelet activation on (A) PMMA nanofibrous mat (white dots indicate the platelet adhesion on nanofibrous mat) and (B) CBMA nanofiber membranes, respectively, (C) blood clotting efficiency of mats using OD values.
hematoxilin and eosin sections indicated the presence of densely packed keratinocytes in the epidermis on wounds treated with the CBMA composite mat, as compared to the bare wound (Fig. 7). The wounds covered with CBMA membranes showed significant vascularization after 3 weeks, and the thickness of the granulation layer was almost comparable to that of nonwounded skin, indicating a perfect healing nature [9]. The CBMA composite mat improved the rate of healing in the treated wounds, as evidenced in the H&E-stained images. The wounds treated with CBMA composite mat showed granular tissue formation and the presence of wide spread glandular cavity. Furthermore, the
formation of thickened epidermis and the keratinocyte restoration along with big rete pegs were also seen in the H&E-stained images. From the H&E-stained images, it is clear that the CBMA mat promotes complete wound healing. The cell attachment to the CBMA composite wound dressing mat was minimized due to the inert cell nature, and hence, skin re-epithelialization was favored since the composite mats did not interact with the cells. The zwitterionic composite mats will provide a favorable environment for healing in this respect [35], and new connective tissue formed after 3 weeks, which is evidence of complete healing when the wound material is used.
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Fig. 5. Antibacterial activity of (A) CBMA nanofibers and (B) control PMMA nanofibers against (1) gram-positive bacteria Staphylococcus aureus and (2) gram-negative Escherichia coli.
Fig. 6. Photographs of the in vivo wound healing study. The extent of wound closure in the wounds treated with CBMA composite mat compared to bare wound are shown in graphs.
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Fig. 7. Photomicrographs of hematoxilin and eosin (H&E)-stained (A and C) bare wound, (B and D) CBMA zwitterionic mat treated wounds. The newly formed epithelium invaded into the granulation tissue to form rete pegs (the triangle, arrow, star, quadrangle, and circle indicate epidermis, rete pegs, granular tissue, glands, and blood vessels, respectively).
4. Conclusions In this work, we successfully fabricated zwitterionic CBMA nanofibers and applied them as wound dressings. The CBMA nanofiber membrane exhibited a superior resistance to cell attachment, platelet adhesion and improved antibacterial activity. The inert cell property of our nanofibrous mats offers many advantages for wound care management, and such non-cell adherent wound dressing membranes can be used as easily removable, painless bandages. These membranes will not cause any pain upon frequent removal. Furthermore, the healing tissue will not be damaged since the newly formed layer of skin is not disturbed. A minimal scar can be expected when such wound dressings are used. We used an in vivo model to prove that incisional wounds take a far longer time to heal in the absence of the CBMA nanofiber membrane. All of the results indicate great potential to use this nonfouling zwitterionic nanofibrous material as a nonstick smart wound dressing material. Acknowledgments This research was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (Project nos. 2013-012911, 2015-020449 and 2015R1C1A1A02036404). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2015.11.086. References [1] J.S. Boateng, K.H. Matthews, H.N.E. Stevens, G.M. Eccleston, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci.-Us 97 (2008) 2892–2923.
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Contents lists available at ScienceDirect
Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Electrospun zwitterionic nanofibers with in situ decelerated epithelialization property for non-adherent and easy removable wound dressing application Afeesh Rajan Unnithan a,b,1, Amin Ghavami Nejad a,1, Arathyram Ramachandra Kurup Sasikala a, Reju George Thomas c, Yong Yeon Jeong c, Priya Murugesan d, Saeed Nasseri d, Dongmei Wu d, Chan Hee Park a,b,⇑, Cheol Sang Kim a,b,⇑ a
Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju, Republic of Korea Division of Mechanical Design Engineering, Chonbuk National University, Jeonju, Republic of Korea c Department of Radiology, Chonnam National University, Hwasun Hospital, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea d Department of BIN Fusion Technology, Chonbuk National University, Jeonju, Republic of Korea b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Non cell adherent wound dressing
membranes. Easy removable, no-pain wound
dressing bandages. Blood inert wound dressing
membrane, suitable for large areas of chronic wounds. Resist microbial biofilm formation and hence provides minimum chance of infection. Minimum cosmetic scar due to less cell adsorption on wound dressing membrane.
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 6 November 2015 Accepted 25 November 2015 Available online 28 November 2015 Keywords: Zwitterion Carboxybetaine Electrospinning Wound dressing Nanomembrane Electrospinning
a b s t r a c t Wound care management is a serious issue among the medical practitioners due to its varying complexity and various materials were tested for fast relief and easy removal. In this regard zwitterionic polymer based wound dressing membranes are the key point of attraction. Here we prepared a novel zwitterionic poly (carboxybetaine-co-methyl methacrylate) (CBMA) copolymer based nanomembranes using the electrospinning technique for the wound dressing application. The study takes advantage of the outstanding chemical properties of zwitterionic CBMA and the morphological efficiency of nanomembranes. The cell attachment studies proved the cell inert nature of thus prepared membranes. Such non cell adherent wound dressing membranes can be applied as the easy removable, no-pain wound dressing bandages. Our results clearly showed that the excellent blood-inert nature can be achieved by the CBMA nanofiber membranes. Therefore, there will be less chance of attaching blood clot with the wound dressing membrane and is extremely significant for the care of patients with large areas of chronic wounds. Additionally the in vivo results showed the formation of new tissues within two weeks, evidence of a complete wound healing material. So our CBMA membrane can be successfully used as a perfect wound dressing material with minimum cosmetic scar. Ó 2015 Elsevier B.V. All rights reserved.
⇑ Corresponding authors at: Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju, Republic of Korea. 1
E-mail addresses: [email protected] (A.R. Unnithan), [email protected] (C.H. Park), [email protected] (C.S. Kim). These authors contributed equally.
http://dx.doi.org/10.1016/j.cej.2015.11.086 1385-8947/Ó 2015 Elsevier B.V. All rights reserved.
A.R. Unnithan et al. / Chemical Engineering Journal 287 (2016) 640–648
1. Introduction The skin is considered to be the largest organ of the body, and is serves many different functions. Therefore, wound care management is a serious healthcare issue. Wound dressings are primarily responsible for managing the most types of open wounds [1]. The immediate care of skin wounds is important to prevent microbial infections as well as trans-epidermal water loss that may cause acceleration in the wound regeneration [2]. Therefore, the restoration of the skin barrier is of crucial importance for the treatment of injuries [3]. Wound dressing materials made via electrospinning technique have been increasingly studied due to their superior properties, and the electrospun nanofibers have been appropriate for use as perfect wound dressing materials as a result of their useful properties, including flexible pore-size distribution, high porosity to provide oxygen permeability, and high surface-to-volume ratio [4–6]. A variety of materials have been electrospun to control the microenvironment of the wound surface as well as to stimulate the healing process, and currently nonadherent, wound dressing materials have been preferred in the sense that they can be easily removed without trauma, possesses antimicrobial properties and promote wound healing with minimal scarring [7–10]. Natural biopolymers, such as chitosan [11,12], alginate [13], cellulose acetate [14] and, hyaluronic acid [15] have been extensively studied for their potential role as materials for wound dressings. The use of such biopolymers is sometimes limited due to their dissimilarities from batch to batch of raw materials and the possibility of the transmission of an infection due to their bioactive nature. Many synthetic polymers, such as polyurethane [10,14], poly (e-caprolactone), and poly (L-lactide) [16], have also been used as wound dressings. The mechanical properties, desirable cytocompatibility and low cost of the synthetic polymers affirm that these are perfect materials for use in wound dressing applications [17]. However, most polymeric materials lack resistance to the nonspecific adsorption of proteins. This can result in nonspecific cell attachment and in bacterial adhesion, which in turn would cause bacterial infections and pain upon removing the wound dressing material as a result of the cell growth over the material [18]. Recent studies have shown that a zwitterionic copolymer composed of carboxybetaine methcrylate monomer exhibits excellent blood compatibility, including the suppression of platelet adhesion and complement activation on the surface of the polymer [19]. They are also known to be resistant to cell adhesion, both in vitro and in vivo [20]. The zwitterionic carboxybetaine polymers have also been reported to possess antibacterial properties and resistance to the formation of bacterial films [21,22]. Their desirable properties, such as a strong resistance to protein adsorption, cell attachment, and resistance to bacterial growth, have made carboxybetaine methcrylate polymers promising materials for use as nonadherent wound dressing materials [23]. Such membranes will not damage healing tissue and can be easily applied and removed, causing no pain [18]. Furthermore, electrospinning is an easy, efficient method that can be used to prepare such nanofibrous membranes. The electrospun nanofibers have been found to be very effective when used as wound dressing materials mainly due to their architectural superiority [24]. We synthesized poly (carboxybetaine-co-methyl methacrylate) copolymer (denoted as CBMA throughout this paper). The application of the methylmethacrylate based material as a wound dressing has already been extensively reported [25]. In this work, a fibrous membrane was electrospun from as-prepared CBMA polymer for use as a wound dressing. The main objective of this study is to develop an electrospun nanofibrous membrane from CBMA in order to study its properties when intended for use as active non-
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adherent wound dressings. The cell and platelet adhesion behavior and the in vivo wound healing efficiency of the zwitterionic CBMA nanofibrous membrane were thus studied. 2. Materials and methods 2.1. Synthesis and characterization of the carboxybetaine monomer [CBAA-5-ester: (6-carboxypentyl)-3-acrylamidopropyl dimethylammonium bromide, ethyl ester] The carboxybetaine monomer was prepared and characterized following methods that were previously reported [26]. N-(3Dimethylaminopropyl) acrylamide (50 mmol), ethyl 6-bromohexanoate (55 mmol), and acetonitrile (25 mL) were added into a 100-mL round-bottom flask. The mixture was stirred under a nitrogen atmosphere for five days at 45 °C. The solvent was removed on a rotary evaporator, and the product was then precipitated and washed with anhydrous ether. In the next step, 100 mg/mL of monomers were dissolved into aqueous solutions with 1 M NaOH concentrations. After 24 h of hydrolysis, the solution was neutralized with a dilute HCl solution and water was then removed by vacuum. 1H-NMR (400 MHz, D2O): 2.00 (m, 4H, CACH2AC), 2.47 (t, 2H, CH2AC@O), 3.1 (s, 6H, N+(CH3) 2, 3.3–3.4 (6H, CHAN and CH2AN + ACH2), 5.75 (m, 1H, CH@CACON-trans), 6.19 (m, 1H, CH@CACON-cis), 6.26 (m, 1H,@CHACONA). 2.2. Synthesis of poly (carboxybetaine -co-methyl methacrylate) copolymer abbreviated as CBMA An appropriate amount of two monomers, MMA (47 mmol) and carboxybetaine monomer (4.7 mmol), were added to a 50-mL round bottomed flask that contained 30 mL of dimethylformamide. The solution was bubbled with nitrogen for 10 min, azobisisobutyronitrile was then added to the flask, and the solution was heated up to 70 °C and was stirred overnight. Next, the solution was added drop-wise to 400 mL of diethyl ether while stirring to precipitate the synthesized copolymer. The purified copolymer was dried overnight in a vacuum oven at room temperature. The final products present a white to light yellow color and had a yield of 68%. The molar ratio of carboxybetaine to MMA in the obtained copolymer was estimated to be 9.8% by 1H-NMR analysis (Fig. 1 supporting information) [27,28] by taking the integral of a peak at 3.4 ppm belongs to the six protons present in the two methyl groups attached to the quaternary amine ((CH3)2N+) of carboxybetaine pendant group, and a peak at 3.7 ppm belongs to the three protons present in the terminal methyl group of the MMA pendant group. It should be mention that deuterated chloroform was used to dissolve copolymer and which results in the sharp peak at 7.24 ppm. The materials were also tested chromatographically, but due to the poor solubility of the materials, it was not possible to provide exact values, even with the addition of salts such as LiCl [29]. The carbobetaine repeat unit of the copolymer is hydrophilic, and makes the polymer insoluble in the relatively hydrophobic solvents [29]. However, since the polymer solution is spinable it can be said the molar masses are high enough for the electrospinning process. 2.3. Fabrication of the CBMA nanofibers Copolymer solutions were prepared by dispersing the polymer at concentration of 20 wt% in dimethyl formamide (DMF). The obtained solutions were placed in a plastic syringe tube and were fed through a metal capillary (nozzle) with a diameter di = 0.21 mm (21 G) attached to a 1-D robot system that moves laterally and is controlled by the LabVIEW 9.0 software program
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(National Instruments). A controllable syringe pump was used to maintain the feeding rate at 0.5 ml/h. Electrospinning was carried out at a voltage of 18 kV and a working distance of 15 cm at room temperature. 2.4. Rheological characteristics The mechanical properties of the polymeric solution were tested using a rheometer (Malvern KinexusPro) with a 20 mm/1° or 2° cone/plate geometry. 2.5. Characterization of the nanofibers Field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan) was used to observe the morphology of the electrospun mats. 2.6. Platelet activation study Blood clotting studies were conducted according to methods previously reported in the literature [30]. Blood was drawn from a voluntary human ulnar vein using a BD Discardit II sterile syringe and was mixed with anticoagulant agent acid citrate dextrose at a ratio of 9:1. Triplicate samples were used for this study, and a pristine PMMA mat was used as the negative control. Blood was added to each of the composite nanofiber mats and was placed in a 25-mL plastic Petri dish, followed by the addition of 10 lL of 0.2 M CaCl2 solutions to initiate blood clotting. These mats were then incubated at 37 °C for 10 min. 15 mL of distilled water were then added drop-wise without disturbing the clot. Subsequently, 10 mL of solution were taken from the dishes and were centrifuged at 1000 rpm for 1 min. The supernatant for each sample was collected and was kept at 37 °C for 1 h. Two hundred microliters (200 lL) of this solution were transferred to a 96-well plate. The optical density at 530 nm was measured using a plate reader (Dynex Technologies USA). For the platelet activation studies, blood was mixed with anticoagulant and was centrifuged at 2500 rpm for 10 min. The supernatant rich in platelet plasma was collected and was added directly to the nanofibers. The samples were fixed with glutaraldehyde and were then washed with PBS. Later these samples were dehydrated using a gradient alcohol treatment and were then viewed under SEM. 2.7. Antibacterial activity measurement with a disc diffusion test All of the bacteria used in this study were purchased from a Korean type culture collection. Escherichia coli (KACC 14818) and Staphylococcus aureus (KACC 10768) were obtained, and all cultures were maintained in brain heart infusion agar (BHIA) plates and were stored at 4 °C. An inhibitory test of CBMA nanofibers was conducted by using the disc diffusion method [31]. The CBMA nanofibrous mats and the pristine PMMA nanofibers were cut into small circular discs with a diameter of around 5 mm each and were denoted as A and B, respectively. These discs were then placed on the surfaces of the petri dishes. The inhibition zones were estimated after 0–240 min. CBMA- and PMMA-only discs were placed as control discs on the plates seeded with the test organisms. The antibacterial activity plates were then incubated at 37 °C, and the diameters of the inhibition zones were measured using a transparent ruler. 2.8. Biocompatibility study The CBMA nanofibers were seeded with 5 104 cells of the NIH3T3 cell line (mouse embryonic fibroblast cells) into 24-well plates and were cultured in DMEM medium in a 5% CO2 incubator
with a humidified environment at 37 °C. The measurements for the nanofiber were taken in triplicate along with the control samples that had cells seeded onto a coverslip (SPL Lifesciences, Korea) only. After 2nd, 4th and 6th day, 50 ll of MTS reagent (Promega, USA) were added to each of the wells and were incubated for 4 h. A microplate reader was used to measure the absorbance at 490 nm. For the cell attachment study, the scaffolds were rinsed twice with PBS and were subsequently fixed in 4% paraformaldehyde after the 2nd and 4th days of incubation. Next, the samples were rinsed with distilled water and were then dehydrated with a graded concentration of ethanol, i.e., 20%, 30%, 50%, 70% and 100% ethanol for 10 min each. Finally, the samples were kept in a vacuum oven and were then sputter-coated with gold to observe the cell morphology by using SEM. After 48 h of incubation, the cells were treated with cytoskeletal phalloidin staining with 200 ll of phalloidin stain (Molecular probes, USA) and DAPI dissolved in PBS for 30 min at room temperature. The coverslip containing the cells was viewed directly under a fluorescent microscope. 2.9. In vivo wound dressing study The in vivo animal study was approved by the Institutional Animal Ethical Committee of Chonbuk National University, Jeonju, South Korea (IACUC certification number CBNU 2014-00062). Wistar rats weighing 200–250 g and aged 4–6 weeks were used in this study. The rats were divided into two groups, with each group containing three rats (n = 3). The rats were allowed to take normal rat feed and water without restriction. On the day of the wounding, the rats were anaesthetized with an intraperitoneal injection of 80 mg/kg ketamine and 8 mg/kg xylazine. The dorsal area of the rats was depilated, and the operative area of the skin was cleaned with alcohol. A partial thickness skin wound with a size of 1.5 cm2 was prepared by excising the dorsum of the rat using surgical scissors and forceps. The prepared wounds were then covered with the CBMA nanofibers, and the rats with the bare wound were covered using cotton gauze. After the dressing materials were applied, the rats were individually housed in cages under normal room temperature. The dressing materials were changed at weeks 1, 2, and 3. During the change in the dressings the hair of the animals was cropped, and photographs were taken. The wound area was then measured using a transparent polyethylene sheet. The sheet was kept on top of the wound, and the area was marked using a marker pen. After week 3, the skin wound tissue of the rats was excised, fixed with 10% formalin, and stained with a hematoxylin eosin (H&E) reagent to conduct histological observations. 3. Results and discussion 3.1. Physical characterizations of composite nanofibers The FESEM images of electrospun CBMA nanofibers are shown in Fig. 1. These randomly oriented nanofibers exhibited a beadfree, smooth surface with almost uniform diameters along their lengths. The diameters of these composite nanofibers were determined to be in the range from 800 to 900 nm. The composite electrospun nanofiber mats produced in this study were desirably smooth and flexible and were successfully used in the desired studies. The porosity measurements using mercury intrusion method also confirmed the porous nature of the CBMA nanofiber mat (Fig. 3 supporting information). Thus, the electrospun CBMA nanofibrous scaffolds possess a perfect porous morphological appearance that is crucial for gaseous and fluid exchange in a wound environment. The water contact angle measurement showed the hydrophobic nature of the CBMA nanofiber mat. The
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Fig. 1. FESEM images of electrospun (A) and (B) CBMA nanofiber mat at lower and higher magnifications respectively.
PMMA nanofiber mat showed a water contact angle of 126.32° ± 2.5°, whereas CBMA mat showed 123.32° ± 1.8° as contact angle. A very small decrease in contact angle was observed in CBMA mat compared to PMMA nanofiber mat, even though both maintained the hydrophobic nature (Fig. 4 supporting information). 3.2. Cell attachment and biocompatibility study A critical property of wound dressing materials is their ability to resist the adhesion of cells instituting epidermis. The adhesion of fibroblasts to the wound dressing material delays healing, which results in an associated supplementary trauma. To observe the cell adhesion and repulsion, the morphological appearances of the cells on the composite nanofiber mats was obtained after 2 and 4 days of culture. Cell attachment studies were conducted using FESEM images (Fig. 2), and these revealed that the cells were attached to the electrospun PMMA nanofibrous scaffolds. In the case of the electrospun PMMA nanofibrous scaffolds, the cells began to spread on the nanofibers by the 4th day. The electrospun PMMA nanofibrous scaffolds served as a good environment for cell attachment and proliferation since they mimic the native extracellular matrix (ECM) morphology and have high area-to-volume ratio and porosity. On the other hand, the electrospun CBMA composite nanofibrous scaffolds showed a significantly low affinity for cell attachment. After 4 days of culture, the cells become rounded and were not able to spread on the surface of the electrospun zwitterionic CBMA nanofibrous scaffold (Fig. 2D). The MTT results also confirmed a lack of cell adhesion to the CBMA nanofibrous scaffold (Fig. 2E). The CBMA nanofibrous surfaces were devoid of any cell attachment proteins as a result of the zwitterionic content, and hence, the cells could not spread onto the CBMA composite nanofibrous scaffolds [32,33]. The improved resistance to cell adhesion was further confirmed via DAPI staining and confocal imaging. The nuclear stain DAPI showed a reduced cell growth in the CBMA nanofibrous scaffold relative to that of pure PMMA nanofibers, as shown in Fig. 3. Phalloidin staining was carried out to understand the response of the cells to the zwitterionic nanofibrous scaffold after 2 days of culturing, and the F-actin stains showed a cytoplasmic filamentous distribution under a confocal microscope. The cell density of the cells that proliferated in the control sample was higher than that in the CBMA nanofiber scaffold. We found that the cell density and morphology were better for the control sample than for the electrospun zwitterionic CBMA nanofibrous scaffolds (Fig. 3A and B). The cells attached to the control PMMA scaffolds and showed sufficient cell-to-cell communication. However, the cells that attached on CBMA nanofibrous scaffolds showed limited
cell–cell communication with a limited distribution of cells. As a result, the cell density was lower on the zwitterionic nanofibrous scaffold than that of the cells that proliferated on the control PMMA scaffold. Zwitterionic membranes are well known to be resistant to protein adsorption and, hence, repel cell adhesion [18,34]. The inert cell property of the nanofibrous mats can be an advantage for wound care management in many ways. Such non cell-adherent wound dressing membranes can be applied as easily removable, painless bandages [35]. These membranes will not cause any pain upon frequent removal, moreover the healing tissue will not be damaged [35,36]. Since the newly formed skin layer is not disturbed, a minimal scar can be expected when such wound dressings are used. 3.3. Platelet activation study The blood inert nature of the CBMA nanofiber mats was studied by conducting a platelet activation analysis. The SEM images showed that the PMMA nanofiber mats allowed the platelets to settle on the nanofiber surface, as seen in Fig. 4A. The SEM images showed that the platelets were spread all over the PMMA nanofibrous mat surface. The SEM results showed the full-scale platelet adhesion and activation at the blood contact site. It is clear that very few platelet attachments from whole blood were observed on the surface of the CBMA nanofiber mats when compared to the full-scale attachment and aggregation of platelets on the control nanofiber mats (Fig. 4B). The optical density measurement revealed that the CBMA nanofiber mats exhibited limited blood clotting ability when compared with pristine PMMA mats, as shown in Fig. 4C. Zwitterionic polymers were already known to have a blood inert nature [37,38], and these results clearly indicate that an excellent blood-inert nature can be achieved by CBMA nanofiber mats, which are shown in Fig. 4B to resist platelet adhesion. The electrostatic attraction between the charges on the pendant groups may contribute to the protein resistance of the CBMA nanofiber membranes. Preferably, the wound dressings should not activate blood defense systems or blood cells, and furthermore, if the surface of the wound dressing membrane interacts with any of the blood components, the wound surface will be at risk once the dressing is removed. Therefore, the probability that a blood clot attaches to the wound dressing membrane is reduced, and hence, non-adherent wound dressings are extremely important for the care of patients with large-area chronic wounds. 3.4. Antibacterial effects The prevention of a microbial infection is a key factor for any type of wound dressing material [39]. The CBMA nanofiber
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Fig. 2. FESEM images showing the cell attachment and morphology on PMMA nanofiber mats and CBMA nanofiber mats after day 2 (A and B) and day 4 (C and D) respectively, (E) MTT cell growth measurement assay. The viability of control cells was set at 100%, and viability relative to the control was expressed.
membranes exhibited a clear zone of inhibition with both Grampositive and Gram-negative bacterial strains. As shown in Fig. 5, the Gram-positive bacteria S. aureus presented a zone of inhibition with a mean diameter of around 15 mm, and the Gram-negative E. coli had an inhibition zone of nearly 18 mm. No bactericidal activity was detected for the pristine PMMA nanofibrous mats. The CBMA nanofiber membranes exhibited excellent bactericidal activity against a wide range of bacteria; therefore exogenous infections were effectively avoided. We observed a small zone of inhibition which might be due to the small molecular quaternary ammonium compounds (QMCs) with carboxybetaine esters. These QMCs could bind to the outer membrane and cytoplasmic membrane of bacteria and permeate into bacterial membranes [26]. The sanitization of exogenous organisms is a critical factor for an ideal wound dressing material. The physicochemical properties of the wound dressing material surface determine the adhesion of the bacteria to the materials. The zwitterionic content is already known to cause microbial resistance and so contributes to the microbial resistance in the CBMA nanofiber membranes. Thus, the CBMA nanofiber membranes that were prepared could be sued to combat biofilm formation, which can potentially affect most wound dressing materials [40]. It is crucial to do so in order to stop or reduce a bacterial infection in its initial stages, which can be life threatening. Therefore, the main advantage of using a CBMA
nanofiber membrane dressing is that it will not cause any fouling in the dressing due to the formation of the microbial biofilm. Hence, it provides a minimum chance of introducing new bacteria with repeated exposure of the wound site to the outside and also provides more comfort to the patients [41]. 3.5. In vivo wound dressing study The main purpose of developing the CBMA nanofiber membranes was to evaluate the skin tissue reconstruction in the wounds. An in vivo study was conducted with Wistar rats, and the wound healing ability of the prepared CBMA nanofibrous composite mat was proven. Fig. 6 shows photographs of the in vivo wound healing study. The CBMA composite mats achieved excellent healing in the first and second weeks relative to the bare wound. The control seemed to delay the wound recovery when compared to the CBMA mat dressings. The extent of the wound closure was evaluated and observed after 3 weeks, and the wounds that were treated with the CBMA mat significantly improved the closure at 92% when compared to bare wounds that showed a wound closure of 70% (Fig. 6). After 3 weeks of contact with the CBMA composite mat, the thickness of the granulation layer was similar to that of unwounded skin, indicating optimal healing. Besides that the qualitative histomorphology in routinely-stained
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Fig. 3. Fluorescent microscopic images of phalloidin staining of cells on (A) control nanofibers, (B) CBMA nanofibers respectively.
Fig. 4. SEM images of platelet activation on (A) PMMA nanofibrous mat (white dots indicate the platelet adhesion on nanofibrous mat) and (B) CBMA nanofiber membranes, respectively, (C) blood clotting efficiency of mats using OD values.
hematoxilin and eosin sections indicated the presence of densely packed keratinocytes in the epidermis on wounds treated with the CBMA composite mat, as compared to the bare wound (Fig. 7). The wounds covered with CBMA membranes showed significant vascularization after 3 weeks, and the thickness of the granulation layer was almost comparable to that of nonwounded skin, indicating a perfect healing nature [9]. The CBMA composite mat improved the rate of healing in the treated wounds, as evidenced in the H&E-stained images. The wounds treated with CBMA composite mat showed granular tissue formation and the presence of wide spread glandular cavity. Furthermore, the
formation of thickened epidermis and the keratinocyte restoration along with big rete pegs were also seen in the H&E-stained images. From the H&E-stained images, it is clear that the CBMA mat promotes complete wound healing. The cell attachment to the CBMA composite wound dressing mat was minimized due to the inert cell nature, and hence, skin re-epithelialization was favored since the composite mats did not interact with the cells. The zwitterionic composite mats will provide a favorable environment for healing in this respect [35], and new connective tissue formed after 3 weeks, which is evidence of complete healing when the wound material is used.
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Fig. 5. Antibacterial activity of (A) CBMA nanofibers and (B) control PMMA nanofibers against (1) gram-positive bacteria Staphylococcus aureus and (2) gram-negative Escherichia coli.
Fig. 6. Photographs of the in vivo wound healing study. The extent of wound closure in the wounds treated with CBMA composite mat compared to bare wound are shown in graphs.
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Fig. 7. Photomicrographs of hematoxilin and eosin (H&E)-stained (A and C) bare wound, (B and D) CBMA zwitterionic mat treated wounds. The newly formed epithelium invaded into the granulation tissue to form rete pegs (the triangle, arrow, star, quadrangle, and circle indicate epidermis, rete pegs, granular tissue, glands, and blood vessels, respectively).
4. Conclusions In this work, we successfully fabricated zwitterionic CBMA nanofibers and applied them as wound dressings. The CBMA nanofiber membrane exhibited a superior resistance to cell attachment, platelet adhesion and improved antibacterial activity. The inert cell property of our nanofibrous mats offers many advantages for wound care management, and such non-cell adherent wound dressing membranes can be used as easily removable, painless bandages. These membranes will not cause any pain upon frequent removal. Furthermore, the healing tissue will not be damaged since the newly formed layer of skin is not disturbed. A minimal scar can be expected when such wound dressings are used. We used an in vivo model to prove that incisional wounds take a far longer time to heal in the absence of the CBMA nanofiber membrane. All of the results indicate great potential to use this nonfouling zwitterionic nanofibrous material as a nonstick smart wound dressing material. Acknowledgments This research was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (Project nos. 2013-012911, 2015-020449 and 2015R1C1A1A02036404). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2015.11.086. References [1] J.S. Boateng, K.H. Matthews, H.N.E. Stevens, G.M. Eccleston, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci.-Us 97 (2008) 2892–2923.
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