Materials Science and Engineering C 44 (2014) 52–57

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Electrospun propolis/polyurethane composite nanofibers for biomedical applications Jeong In Kim a, Hem Raj Pant a,b,c,⁎, Hyun-Jaung Sim d, Kang Min Lee e, Cheol Sang Kim a,⁎⁎ a

Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561–756, Republic of Korea Department of Engineering Science and Humanities, Pulchowk Campus, Tribhuvan University, Kathmandu, Nepal Research Institute for Next Generation, Kalanki, Kathmandu, Nepal d Department of Bioactive Material Science, Research Center of Bioactive Material, Chonbuk National University, Jeonju, Chonbuk, Republic of Korea e Department of Molecular Biology, College of Natural Science, Chonbuk National University, Jeonju, 561–756, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 20 March 2014 Received in revised form 9 July 2014 Accepted 25 July 2014 Available online 2 August 2014 Keywords: Propolis Polyurethane Nanofibers Biomaterials Electrospinning

a b s t r a c t Tissue engineering requires functional polymeric membrane for adequate space for cell migration and attachment within the nanostructure. Therefore, biocompatible propolis loaded polyurethane (propolis/PU) nanofibers were successfully prepared using electrospinning of propolis/PU blend solution. Here, composite nanofibers were subjected to detailed analysis using electron microscopy, FT-IR spectroscopy, thermal gravimetric analysis (TGA), and mechanical properties and water contact angle measurement. FE-SEM images revealed that the composite nanofibers became point-bonded with increasing amounts of propolis in the blend due to its adhesive properties. Incorporation of small amount of propolis through PU matrix could improve the hydrophilicity and mechanical strength of the fibrous membrane. In order to assay the cytocompatibility and cell behavior on the composite scaffolds, fibroblast cells were seeded on the matrix. Results suggest that the incorporation of propolis into PU fibers could increase its cell compatibility. Moreover, composite nanofibers have effective antibacterial activity. Therefore, as-synthesized nanocomposite fibrous mat has great potentiality in wound dressing and skin tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Propolis (bee glue) is a resinous substance produced by bees from plant exudates, beeswax, and their salivary secretions. The bees use propolis as a sealer for cracks and crevices of the hive [1,2]. Its composition varies according to the local flora, climate conditions, season and other constituents such as wax, pollen, organic compounds [3]. Propolis contains a great variety of chemical compounds mainly flavonoid aglycones, phenolic acids and their ester derivatives, phenolic aldehyde, ketons and alcohols, steroids, amino acids and some inorganic compounds [4–6]. Its color varies from green, red, to dark brown, and it represents highly adhesive properties [6]. In different part of the world, some of the beekeeper has discarded propolis as a byproduct of honey industry. However, in some communities, it is highly appreciated for its extraordinary properties. Different investigations indicate that the propolis has good medicinal and therapeutic values. Antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory, antitumor and many more properties of propolis

⁎ Correspondence to: H.R. Pant, Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561–756, Republic of Korea. Tel.: +82 63 270 4284; fax: +82 63 270 2460. ⁎⁎ Corresponding author. Tel.: +82 63 270 4284; fax: +82 63 270 2460. E-mail addresses: [email protected] (H.R. Pant), [email protected] (C.S. Kim).

http://dx.doi.org/10.1016/j.msec.2014.07.062 0928-4931/© 2014 Elsevier B.V. All rights reserved.

were reported [7,8]. Considering its unique adhesive and these medicinal properties, the present study was aimed to incorporate propolis through electrospun polyurethane (PU) nanofibers for different biomedical applications. Electrospinning is a versatile technique producing non-woven polymeric membranes containing nanofibers (diameter ranging from few nanometers to hundreds of nanometer) [9,10]. These nanofibers possess the characteristic features of sufficient surface areas and porosity, enabling it to be applied for adsorption, filter, cloths, catalytic support, and tissue scaffold [11–17]. Tissue scaffold is a support that mimics the extra cellular matrix (ECM) and serves as a temporary skeleton for cell growth, migration, and finally reproduction to allow tissue regeneration. Therefore, fabrication of nanofibrous matrices with interconnected porous networks with large void volumes and high surface-to-volume ratios could provide adequate space for cell migration and attachment within the structure. The function of polymeric nanofiber membrane can be improved by incorporating different active components through it. Therefore, the PU (an FDA approved polymer) composite nanofibers that integrates the favorable properties of propolis is expected to significantly improve in material properties for biomedical application. In this report, a facile way for propolis/PU composite nanofiber from simple blending was successfully reported. The adhesive property of propolis provides the point-bonding to the PU fibers and enhances

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Fig. 1. FE-SEM images of PU fibers obtained from (a) 0, (b) 5, (c) 10, and (d) 30 wt.% propolis containing PU solution and insets are their corresponding water contact angle.

their mechanical properties. Moreover, active constituents of propolis on the surface of fibers introduce additional functionalities to the PU fibers. Since many researchers have reported that PU has frequently used for wounds dressing because of its oxygen permeability and excellent barrier properties, the incorporation of propolis through PU fibers further makes it potential in this field [18,19]. So far, no composite bio-membrane of electrospun PU or other polymeric fibers containing propolis has been reported. The antibacterial activity of propolis/PU composite fibers was evaluated using gram negative bacteria (Escherichia coli). The cytocompatibility of as-fabricated composite PU nanofibers with different amounts of propolis was studied using MTT test. The results showed that the incorporation of

small amounts of propolis through PU fibers could enhance some physicochemical properties of PU fibers. These improved properties of PU mat caused by propolis could make it a potential candidate in wound dressing and skin tissue engineering.

2. Experimental 2.1. Materials Propolis (Homart, Australia) and polyurethane pellets (Skythane X595A-11) were used to make propolis/PU electrospun mats.

Fig. 2. FT-IR spectra of different electrospun mats (inset is FT-IR spectra of propolis) where right hand side figure shows the increasing peak intensity at 1638 cm−1 with increasing amount of propolis.

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Tetrahydrofuran (THF) and N,N dimethylformamide (DMF) purchased from Showa (Japan) were used as solvent. 2.2. Electrospinning In this experiment, different PU mats were electrospun from 10 wt.% PU solution containing 0, 5, 10, and 30 wt.% propolis with respect to PU solution. 10 wt.% PU solution was prepared by dissolving PU pellets in mixed solvent system containing 1:1 (by wt) of THF/DMF followed by drop wise addition of propolis with continuous magnetic stirring. Electrospinning was carried out at room condition where the parameters include 17 kV applied voltage, tip-to-collector distance of 14 cm, and solution feed rate of 0.5 ml/h [20]. The electrospun fibers were collected on the surface of rotating dorm (9 cm diameter) using polyethylene sheet. After electrospinning, the nanofibrous mat was dried in vacuum for 12 h at 25 °C. 2.3. Characterizations The fiber morphology of the electrospun nanofibers were observed using field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan) and scanning electron microscopy (SEM, Hitachi S-7400, Hitachi, Japan). From each electrospun mat, about 100 nanofibers were used to measure the average fiber diameter. The presence of propolis in polyurethane fibers was determined using Fourier transform infrared (FT-IR) spectroscopy (ABB Bomen MB100 spectrometer, Bomen, Canada) and thermal gravimetric analysis (TGA) (PerkinElmer, USA). Mechanical properties measurements were carried out using a universal testing machine (AG-5000G, Shimadzu, Japan) at room temperature. Standard dumbbell-shaped samples of different mats were prepared according to the procedures of ASTM D-638 via die cutting from the electrospun mats. The specimen thicknesses were measured using a digital micrometer with a precision of 1 μm. The wettability data of the electrospun nanofibrous mats was determined with deionized water contact angle measurements using a contact angle meter (GBX, Digidrop, France). 2.4. Antibacterial activity test Bacterial viability tests were conducted using a zone inhibition method. Circular discs (diameter = 6 mm) were placed in bacterial suspension in agar plate with an initial Escherichia coli bacteria concentration of 10 5 colony forming unit and incubated up to 24 h at 30 °C. The zones of inhibition were determined by measuring the clear area formed around each disc sample.

Fig. 4. Representative stress–strain curves of PU mats containing (a) 0, (b) 5, (c) 10, and (d) 30 wt.% propolis.

2.5. Cell culture 3T3-L1 fibroblast cell lines with density of 2 × 104 cells/well were seeded on the different electrospun scaffolds. The cells were cultured in minimum essential medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin followed by incubation in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were then grown on the scaffolds for 1 day and 3 days. The nonadherent cells were removed by rinsing with a phosphate buffered saline solution. The adherent cells were fixed with 2.5% glutaraldehyde, dehydrated through a graded series of alcohol (50, 60, 70, 89, 90, and 100%), dried for 12 h in clean bench and morphology was examined by SEM. 2.6. MTT assay The viability of cultured 3T3-L1 fibroblast was monitored on the first, third, and seventh day of culture using MTT (3-[4,-dimethylthiazol-2-yl]2, 5-diphenyltetrazolium bromide; thiazolyl blue) assay. Electrospun scaffolds with same dimension were cut and sterilized under UV radiation for 1 h. The samples were washed three times with sterile PBS prior to transfer to individual 96-well tissue culture plates. Cells were seeded onto the surface of a scaffold, and grown in the α-MEM medium. After incubation at 37 °C in a humidified 5% CO2 incubator for different days, the culture medium was removed and washed twice with sterile PBS followed by the addition of 60 μL of 0.5 mg/ml MTT solution to each well. After 3 h incubation at 37 °C, 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The dissolved solution was swirled homogeneously for about 10 min by the shaker. The optical density of the formazan solution was detected by a 96-well plate reader at 575 nm. The results were expressed as the mean ± standard error of the mean. The data were analyzed via the Student's t test and repeated measures of analyses of variance (ANOVA) test. A probability of less than 0.01 was considered to be statistically significant. 3. Result and discussion 3.1. Physicochemical properties of membranes

Fig. 3. TGA graphs of different electrospun mats.

The morphology of different nanofiber membranes was shown in Fig. 1 (FE-SEM images). For pristine PU electrospun mat (Fig. 1a), the fibers appear uniform and well-defined without any interconnection among the fibers. The composite mats containing different amounts of

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Fig. 5. Zone of inhibition tests for electrospun PU fibers containing (a) 0, (b) 5, (c) 10, and (d) 30 wt.% propolis using E. coli bacteria.

propolis also showed the uniform fiber diameter with slightly changes in fibrous morphology. As we increased the concentration of propolis, clear point-bonded morphology was appeared (Fig. 1d). It is clear that the high propolis concentration provide the connection of two PU fibers because of its adhesive properties [3]. Such point-bonded structures represent the optimum utilization of the bonding element for reinforcement of the nonwoven fabric [21]. It was observed that the increasing amount of propolis not only provided point-bonded structure but also increased the diameter of PU fibers. The measurement on distribution of diameter size of fibers indicated that the fiber diameter was gradually increased with the amount of propolis. The average fiber diameter of PU

Fig. 6. MTT cytotoxicity test on different mats after 1, 3, and 7 days culture. The viability of control cells was set at 100%, and the viability relative to the control was expressed. The data is reported as the mean ± standard deviation (n = 5 and p b 0.05).

fibers containing 0, 5, 10, and 30 wt.% propolis was 204.4, 321.4, 377.2, and 556.6 nm, respectively. Since viscosity of electrospinning solution can play an important role for fiber formation, we have measured the effect of propolis amount on the viscosity of PU solution. It was found that the viscosity of PU solution was gradually decreased with the amount of propolis. The viscosity of PU solution containing 0, 5, 10, and 30 wt.% propolis was 525, 405, 381, and 216 cP, respectively. Therefore, the decreased viscosity of electrospinning solution caused by increasing amount of propolis is responsible to increase the fiber diameter. We expect that the point-bonded structure of composite fibers might be due to the phase separation of two components (PU and propolis) as we observed in our previous work regarding the formation of LA/nylon-6 or chitin buytarite/nylon-6 fibers [20,22]. However, our TEM (figure not shown) observation clearly showed that there was no any phase-separated morphology in composite fibers. It reveled that highly intermisiable composite fiber was formed during electrospinning. Therefore, low propolis containing PU fiber could not give clear point-bonded fibers (Fig. 1b). However, high propolis containing fibers indicated that clear interconnected fibers were formed during electrospining (Fig. 1d). These connection as point-bonded structure is due to the adhesive property of propolis [3]. The formation of composite PU fibers with homogeneous propolis distribution in molecular level was further evaluated by FT-IR spectra. The spectra of PU fibers containing 0, 5, 10, and 30 wt.% propolis are shown in Fig. 2. The typical characteristics PU bands were similar to those of our previous reports [23,24]. In composite fibers, there was no pronounced change in FT-IR spectrum compared to the pristine PU fibers. However, the peak intensity gradually decreased with increasing amount of propolis. It revealed that propolis was homogeneously distributed throughout the PU matrix. Moreover, the characteristic peak of propolis at 1638 cm− 1 (C_O stretching vibration [25]) (see inset of Fig. 2) became stronger with increasing concentration of propolis (right side spectra of Fig. 2). This result indicated that

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Fig. 7. Morphology of fibroblast cells on different mats after 1 day and 3 days cell culture.

sufficient amount of propolis could be easily uploaded through PU fibers during electrospinning. The incorporation of propolis through PU matrix was further evaluated using thermal stability of electrospun composite mats by TGA analysis. Fig. 3 shows the TGA curves of control PU and composite PU mats containing different amount of propolis. For the control PU sample, the initial decomposition temperature is about 288 °C. From the curves, it can be noted that the blended mats display single-stage thermal degradation. The thermal decomposition temperature of composite PU fibers was lowered compared to pristine PU, and it was gradually decreased with increasing amount of propolis (Fig. 3). It may be due to the lower thermal stability of propolis present in PU fibers. Moreover, the proper incorporation of propolis through PU matrix may affect the orientation of polymer chain and cause the decrease in crystallinity. Therefore, there is gradual decrease in thermal stability with increasing amount of propolis in the composite. The mechanical strength of electrospun nanofibrous mat is universally acknowledged as an important factor for wound dressing and tissue scaffolds. The tensile strength of pristine and composite PU fibrous mats is shown in Fig. 4. It revealed that composite PU mat having 5 wt.% propolis has the highest tensile strength. The tensile strength of higher propolis (10 and 30 wt.%) containing PU mats is less than that of 5 wt.% propolis containing mat. This result revealed that 5 wt.% propolis is the suitable amount for the fabrication of PU/propolis composite fibers. Over this concentration, the propolis hinders the proper orientation of polymer molecules and may cause the decrease in tensile strength. Here, propolis could easily intermix with PU solution and decrease its viscosity with increasing amount of propolis which hinders the proper orientation of PU molecules during solidification. The adhesive properties of propolis might be useful to increase the mechanical strength of PU fibers. The water contact angle measurement is a proper technique to measure the wettability of a membrane surface. Hydrophilic surface is essential for wound dressing and tissue engineering. The increased hydrophilicity of composite PU membrane can make it

more biocompatible for aforementioned applications. The contact angle measurement of pristine PU and composite PU mats are shown in inset of Fig. 1. It reveals that water contact angle of PU membrane is gradually decreased with the increasing amount of propolis. Different functionalities of propolis might be the cause of increased hydrophilicity of composite PU membranes. 3.2. Antibacterial study The antibacterial effect of as-synthesized membranes was evaluated using zone inhibition method at room temperature. The effect of propolis on PU membrane towards bacteria is clearly seen in Fig. 5. Here, the diameter of bacterial inhibition zone is gradually increased with increasing amount of propolis in PU membrane. Definitely PU membrane containing 30 wt.% propolis shows the highest inhibitory effect (i.e., the largest clear area around the sample as compared to the other membranes). Different constituents such as flavonoids and cinnamic acid derivative presented in propolis are responsible for the antibacterial effect of composite mat [25,26]. Higher amount of propolis in PU membrane could release more propolis from the composite membrane. Therefore, the antibacterial activity of composite PU membrane is increased with increasing amount of propolis. 3.3. Biocompatibility in vitro In vitro cell viability is an important technique to evaluate the biocompatibility of different biomaterials. Therefore, the fibroblast cells on different PU membranes were cultured for 1, 3, and 7 days, and then the viabilities of cell were determined by MTT assay. As shown in Fig. 6, the number of cells on composite PU scaffolds is increased with increasing amount of propolis at day 1. However, comparing the cell viability at day 7, 30 wt.% propolis containing PU scaffold showed significant low cell proliferation relative to other scaffolds. This is possibly related to less porous fiber morphology due to the thick composite fibers (Fig. 1d) and the release of excess unfavorable constituents

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of propolis from the composite fibers [27]. The porous structure of tissue scaffold plays an important role for the differentiation and proliferation of cells. Therefore, we conclude that 5 wt.% propolis is a suitable amount to fabricate more biocompatible scaffold. To study the fiber–cell interaction behavior of pristine PU and composite scaffolds, SEM micrographs of fibroblast on different scaffolds was observed after 1 and 3 days of cell culture. Fig. 7 shows that the cell growth is higher on propolis/PU scaffold than on pristine PU scaffold. PU, being a biocompatible and nontoxic synthetic polymer, on blending with propolis provides a scaffold with improved cell affinity for skin tissue engineering. Morphology of cell on fibers showed that the cells were well attached on the surface of composite nanofibers compared to the pristine PU nanofibers. The increased hydrophilicity and adhesive behavior of propolis containing PU fibers might be the possible reason for better proliferation of fibroblast. Therefore, with the antibacterial activity and nontoxicity to the cells, propolis loaded PU membrane will be a potential candidate for wound dressing and skin tissue engineering. 4. Conclusion In summary, functional propolis loaded PU composite nanofibrous scaffold was successfully fabricated using electrospinning process. Continuous smooth nanofibers without any phase-separated morphology were obtained from simple blending of two components. Blending of propolis with PU resulted in improved physiochemical and biological characteristics. Antibacterial activity studies proved that as-synthesized PU composite mat has good antibacterial capacity. In vitro cytocompatibility studies revealed that the composite scaffold showed enhanced cell viability. All these results indicated that composite PU scaffold, compared to the pristine PU scaffold, appeared to have improved properties for wound dressing and skin tissue engineering. Acknowledgement This research was supported by grant from the Basic Science Research Program through National Research Foundation of Korea

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polyurethane composite nanofibers for biomedical applications.

Tissue engineering requires functional polymeric membrane for adequate space for cell migration and attachment within the nanostructure. Therefore, bi...
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