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Ultraporous interweaving electrospun microfibers from PCL–PEO binary blends and their inflammatory responses† a b a Yan-Fang Li,‡ab Marina Rubert,‡a Hu ¨ snu ¨ Aslan, Ying Yu, Kenneth A. Howard, a a a Mingdong Dong, Flemming Besenbacher and Menglin Chen*
Production of one dimensional nanomaterials with secondary morphology exhibiting unique functions is challenging. Here we report for the first time that a nanoscale immiscible polymer blend solution electrojet can assemble into ultraporous interweaving microfibers. This intriguingly novel morphology originated from a blend of polycaprolactone (PCL) and polyethylene oxide (PEO) in a DCM–DMF mixed solution when the ratio between each component reached a threshold and when the electrospinning parameters were delicately controlled. The morphology, crystallinity, surface chemistry and wettabilities were characterized to understand the mechanism of formation. The interplay of the two semi-crystalline polymers and the pair of solvents/non-solvents with the electrospinning processing parameters was found to be critical for the formation of the unique structure. Furthermore, the interesting combination Received 21st November 2013 Accepted 22nd December 2013
of biocompatible, biodegradable PCL with protein-resistant PEO motivated us to assess its inflammation
DOI: 10.1039/c3nr06197c
responses on the RAW 264.7 macrophage cell line. All fibers were found to be biocompatible with low inflammation potential upon incubation, while compared with pure PCL nanofibers; the unique
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interweaving microfibers induced a slightly higher inflammatory reaction.
Introduction Production of one dimensional nanomaterials with secondary morphology exhibiting unique functions is challenging. Electrospinning, one of the most promising nanotechnology innovations, has attracted great attention as a versatile method to produce continuous sub-micrometer-scale bers with large surface area, high porosity, controllable mechanical properties, and ease of functionalization1–4 for a wide range of applications in tissue engineering, drug delivery, ltration, sensors, and so on.5,6 While conventional electrospun bers usually have smooth surfaces without interior structures or specialized architecture, it is possible to develop bers with special structures, such as core–shell,7,8 hollow,9 and porous10–12 that may install material-specic functions for potential applications.13,14 The porosity within electrospun bers, both the surface and interior porosity, due to their increased surface area, is of a
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark. E-mail: [email protected]
b
Institute of Nanoscience and Nanotechnology, Central China Normal University, Wuhan 430079, China
† Electronic supplementary information (ESI) available: High resolution SEM image, AFM image, and TEM image of mat VI, PCL–PEO ¼ 10% : 3%. Morphology transformations of mat V: PCL–PEO ¼ 12% : 2.4% by changing different electrospinning parameters and solvent ratios. Contact angles of PCL–PEO bers before and aer water treatment. See DOI: 10.1039/c3nr06197c ‡ Yan-Fang Li and Marina Rubert contributed equally to this work.
3392 | Nanoscale, 2014, 6, 3392–3402
signicant importance in inuencing the nal application.15 A straightforward approach is using porogens. Poly-L-lactide (PLA) bers with interior porous structures were produced from electrospinning a PLA/PVP (polyvinylpyrrolidone) blend followed by selective removal of PVP in water.16 Interestingly, a delicate choice of polymers and solvents could allow phase separation into polymer rich and poor regions, with the latter becoming the pores. Porous electrospun PLA bers were reported by Bognitzki et al., where the porous structure was induced by the rapid evaporation of the solvent dichloromethane (DCM).10 Megelski et al.11 also investigated electrospun bers with different polymers/solvents with micro- and nanopores on the surface. Much attention has been focused on the development and investigation of binary polymer blends that allow the combination of desirable properties of different polymers with advantages over the synthesized novel polymeric materials.17,18 Many polymers are immiscible from the thermodynamic point of view, because the entropy contribution to Gibbs energy of mixing is negligible. Immiscible blends are particularly intriguing as they oen provide unique properties with special polymer architecture.19–21 Most of the investigated blended systems represent amorphous polymers; only few containing crystalline polymers have been studied, which were found to be complicated as both components are able to crystallize and provide various conned conditions for dening the crystallization behavior and resulting
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morphology.22 Crystallization is a solid–liquid separation process, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. It oen occurs during cooling or solvent evaporation; however crystallization during electrospinning has been scarcely investigated.23 Polycaprolactone (PCL) and polyethylene oxide (PEO) are both semicrystalline and they are immiscible. While the mixing of immiscible components is challenging,24 copolymers consisting of both PCL and PEO segment blocks exhibit a highly asymmetric lamellar structure and spherical or cylindrical structures.25–27 Here, a novel multi-lamellar cylindrical structure was fabricated via electrospinning of PCL–PEO blends directly from their homogeneous solutions in DCM/DMF. The morphology, crystallinity, surface chemistry and wettability of these bers were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD), X-ray photoelectron spectrometry (XPS), and contact angle (CA) measurements. Based on the morphology and structure characterization, the formation mechanism of the novel structure could be proposed. Both PCL and PEO are biocompatible and biodegradable and, thus, the most extensively studied polymers for electrospinning and widely used in tissue engineering28 and drug delivery.29–34 Since PEO is also well known to suppress protein adhesion35 and applied as antiinammatory polymeric coatings for implantable biomaterials and devices,36 the bers with different morphologies were applied on macrophage cell cultures. The toxicity of the different PCL–PEO bers as potential biomaterials was evaluated by quantication of the lactate dehydrogenase (LDH) activity, and the expression of inammation related marker genes was evaluated by real time RT-PCR. The cell attachment and morphology were further assessed by SEM and confocal microscopy.
Materials and methods
DMF (3 : 2) at room temperature and stirring until homogeneous solutions formed. The detailed information on the electrospinning solutions is listed in Table 1. The viscosity of the polymer solution was measured using a rheometer with a cone-plate conguration (Physica MCR 501, Anton Paar). The measuring cone was a CP50-1 with a diameter of 50 mm and an angle of 1 . Temperature of the cone and plate were kept at 20.0 C for all the solutions. The conductivity measurement was conducted using a laboratory conductivity meter (inoLab Cond 720), and the temperature of measuring was 24.1 C. The homogeneous polymer solutions were placed in a 1 mL syringe tted with a metallic needle of 0.9 mm inner diameter. The syringe was xed horizontally on the syringe pump (Model: KDS 101, KD Scientic), an electrode of high voltage power supply (Spellman High Voltage Electronics Corporation, MP Series) was clamped to the metal needle tip, and a grounded stationary rectangular metal collector (15 cm 20 cm) covered by a piece of clean aluminum foil was used for the ber collection. The electrospinning process was carried out under the following conditions: applied voltage ¼ 18 kV, feeding rate ¼ 1 mL h1, and distance between the tip of the needle and collector ¼ 12 cm. The experiments were carried out at room temperature, and the relative humidity was between 30% and 60%. The obtained bers were dried under vacuum (labconco Freezone Triad™) overnight to remove the excess solvents before further use. Scanning electron microscopy The morphologies of the electrospun bers were examined with a high-resolution scanning electron microscope (SEM) (FEI, Nova 600 NanoSEM). The bers were placed directly into the SEM chamber without any metal sputtering or coating; all the images were captured using a secondary electron detector with an acceleration voltage of 5 kV. Atomic force microscopy
Electrospinning The polymer solutions were prepared by dissolving PCL (Mw ¼ 70 000–90 000, Aldrich) and PEO (Mw ¼ 900 000, Aldrich) in DCM/
Aer immobilizing electrospun bers on at substrates the quantitative nanomechanical properties of the samples were characterized using a Dimension FastScan AFM (Bruker, Santa
Table 1 Electrospinning solution concentrations, viscosity, conductivity and the resulted electrospun fiber morphology, surface chemistry and wettability
% PEO Mat
%PCL (w/v)
%PEO (w/v)
Viscosity of solution (Pa s)
Conductivity of solution (ms cm1)
I II III IV V VI VII VIII IX X XI
20 18 16 14 12 10 8 6 4 2 0
0 0.6 1.2 1.8 2.4 3 3.6 4.2 4.8 5.4 6
1.06 1.01 0.79 0.67 1.33 1.98 3.12 6.60 3.35 3.78 3.81
1.9 2.0 2.2 1.8 2.5 2.6 2.9 3.1 3.1 3.5 2.7
a
Morphology
Solution
Surfacea
Contact angle ( )
Nanobers (0.92 0.61 mm) Pores on bers (3.01–3.39 mm)
0 8.08 16.51 25.32 34.52 44.16 54.26 64.86 75.98 87.68 100
0 33.28 39.29 51.18 45.83 58.14 65.65 58.47 64.01 82.67 100
117.9 89.1 84.6 28.8 19.9 23.0 33.1 37.0 34.7 40.3 16.4
Ultraporous interweaving microbers (3.45–4.78 mm) Melded bers Nanobers (0.56 0.11 mm)
% PEO ¼ ½ O–C (PEO)/(½ O–C (PEO) + O–C (PCL)).
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Barbara, CA, USA) under ambient conditions (temperature: 20 C and humidity: 51%). Peak force tapping was performed using triangular silicon nitride probes (nominal resonance frequency: 400 kHz, nominal spring constant: 4 N m1, and tip radius: 5 nm; FastScan-B, Bruker, USA). Topography, stiffness and adhesion images were acquired simultaneously. The linear scanning rate was set to 0.1–0.5 Hz with a scan resolution of 512 per line. The imaging force and other scan parameters were optimized to achieve the highest possible resolution also to avoid damaging the sample or wearing the tip. Collected images were further processed using SPIP soware (Image Metrology ApS, Lyngby, Denmark). Alignment of bers was quantitatively determined using Fast Fourier Transformation (FFT) calculations. Transmission electron microscopy The interior structures of the bers were detected by transmission electron microscopy (TEM) (Tecnai G2 F20 U-TWIN) at 200 kV. All the samples were prepared by electrospinning bers directly on carbon coated copper grids. X-ray diffraction X-ray diffraction measurements were carried out using a D8 Discover X-ray diffractometer with Cu Ka radiation (l ¼ 0.15406 nm) at a scan rate of 0.01 2q s1 for the characterization of the crystal structure of the electrospun bers. X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) was performed using a Kratos Axis UltraDLD instrument equipped with a monochromated Al KR X-ray source (hn ¼ 1486.6 eV) operating at 15 kV and 15 mA (225 W). A hybrid lens mode was employed during analysis (electrostatic and magnetic), with an analysis area of approximately 300 mm 700 mm. For each sample, a takeoff angle (TOA) of 0 (with respect to the sample surface) was used, allowing maximum probe depth (10 nm). Wide energy survey scans (WESSs) were obtained over the range of 0–1200 eV binding energy (BE) at a pass energy of 160 eV and used to determine the surface elemental composition. Contact angle The contact angle (CA) was measured by means of a Kr¨ uss Drop Shape Analysis System DSA100. A water droplet of 3 mL was dropped on the surface of the scaffold each time, and the CA values were recorded by continuous shooting mode with an interval of 5 ps1 for 15 s. Each sample was measured three times at different positions. CA values of bers aer water treatment were also measured in the same way. Before measuring the CA, bers were rstly immersed into Milli-Q water (18.2 MU cm at 25 C) for 48 h, rinsed with a large amount of Milli-Q water and then dried in a freeze drier overnight. Cell culture of RAW 264.7 cells on PCL and PEO–PCL bers The murine RAW 264.7 macrophage-like cell lines (ATCC, Manassas, USA) were routinely cultured in DMEM–glutamax
3394 | Nanoscale, 2014, 6, 3392–3402
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(4.5 g L-D-glucose, () pyruvate) (GIBCO, Grand Island, NY) supplemented with 10% FBS (Biowhittaker, Walkersvile, MD) and antibiotics (100 IU and 100 IU streptomycin antibiotics) (Gibco, Grand Island, NY) at 37 C in a humidied atmosphere of 5% CO2. Cells were routinely subcultured in a 1 : 10 ratio before reaching conuence by scrapping. All experiments were performed aer 17 passages of the RAW 264.7 cells. Before seeding, three groups of bers (PCL, 1.8% PEO–14% PCL, and 3% PEO–10% PCL) of 12 mm Ø (1.1 cm2) were punched out and placed on the bottom of a sterile standard 48well plate, with a PCL ring (11 mm Ø) on the top. PCL rings were also placed into the TCP wells, which served as positive and negative reference groups. Raw 264.7 cells were seeded onto each ber at a density of 1.6 105 cells cm2 and maintained in DMEM–glutamax supplemented with 10% FBS and 1% antibiotics. In parallel, cells seeded on a 48-well TCP plate (() TCP) and TCP plus 0.1 mg mL1 lipopolysaccharide ((+) LPS TCP) (LPS, E. coli 055:B5, Schnelldorf, Germany) served as a negative and positive control, respectively. Cells were maintained under standard cell culture conditions (37 C in a humidied atmosphere of 5% CO2) for 24 hours. Aer 24 hours of cell seeding, the culture medium was collected to evaluate cytotoxicity (LDH activity). Cell attachment and morphology onto the bers was also visualized by SEM and confocal microscopy. In parallel, the expression of marker genes related to inammation was assessed by real-time RTPCR. To ensure that the cell characterization was done only on the cells growing onto the bers, samples were moved to a tube prior to gene expression analysis. Cell adhesion and morphology on PCL and PCL–PEO bers The cell morphology aer 24 hours of seeding was visualized using a confocal microscope (CLSM 700 Zeiss, Jena, Germany) and using a scanning electron microscope (SEM) (FEI, Nova 600 NanoSEM). For confocal microscopy images, RAW 264.7 cells adhered to the bers were xed with 4% formaldehyde in PBS for 15 minutes. For staining, cells were permeabilized in 0.2% Triton in phosphate buffered saline (PBS) (Sigma, Schnelldorf, Germany). The cytoskeleton of the cells was stained using 50 mg mL1 phalloidin Atto 488 (Sigma Aldrich, Schnelldorf, Germany) and the nuclei with Prolong® Gold Antifade Reagent with DAPI (Invitrogen, Carlsbad, CA, USA). SEM imaging was performed aer cell xation with glutaraldehyde 4% in PBS for 2 h followed by a dehydration process with ethanol as previously described.37 Cell viability (lactate dehydrogenase (LDH) activity) Aer 24 hours of cell seeding, the LDH activity in the collected culture media was taken as an indicator of membrane leakage/ cell lysis. The activity of the cytosolic enzyme was estimated according to the manufacturer's kit instructions (Roche Diagnostics, Mannheim, Germany), by assessing the rate of oxidation of NADH at 490 nm in the presence of pyruvate. Aer removing the background in the absorbance of the culture media without cells, results from all the samples were presented
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relative to the LDH activity in the medium of cells treated cultured on tissue culture plastic (TCP) (low control, 0% of cell death) and of cells cultured on TCP treated with 1% Triton X-100 (high control, 100% cell death). The percentage of LDH activity was calculated using the equation:
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Cytotoxicity (%) ¼ (exp. value low control)/ (high control low control) 100.
Total RNA isolation and gene expression of inammation markers by real-time RT-PCR The effect of bers to induce an inammation response was further studied by quantication of relative mRNA levels of selected inammation related markers aer 24 hours of cell seeding. Total RNA was isolated from cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. Total RNA was quantied at 260 nm using a Nanodrop spectrophotometer (IMPLE AH Diagnostics, Helsinki, Finland). 0.4 mg RNA was reverse transcribed to cDNA at 37 C for 60 min using a high capacity RNAto-cDNA kit (Applied Biosystems, Foster City, CA), according to the protocol of the supplier. Aliquots of each cDNA were frozen (20 C) until the PCR reactions were carried out. Real-time PCR was performed in a Lightcycler 480® (Roche Diagnostics, Mannheim, Germany) using SYBR green detection. Real time RT-PCR was done for three reference genes (18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TBP) and 3 target genes (tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b) and nitric oxide synthase (iNOS)). The primer sequences were as follows: 18s rRNA-F: 50 -GTAACCCGTTGAACCCCATT-30 ; 18s rRNA-R: 50 -CCATCCAATCGGTAGTAGCG-30 ; GAPDH-F: 50 -ACCCAGAAGACTGTGGATGG-30 ; GAPDH-R: 50 -CACATTGGG-GGTAGGAACAC-30 ; TBPF: 50 -AGAGAGCCACGGACAACTG-30 ; TBP-R: 50 -ACTCTAGCA TATTTTCTTGCTGCT-30 ; TNFa-F: 50 -GTAGCCCACGTCGTAG CAAAC-30 ; TNFa-R: 50 -ATCGGCTGGCACCACTAGTT-30 ; ILb-F: 50 GCCACCTTTTGACAGTGATGA-30 ; ILb-R: 50 -GATGTGCTGCTGC GAGATTT-30 ; iNOS-R: 50 -GCCACCAACAATGGCAACAT-30 ; iNOSF: 50 -TCGATGCACAACTGGGTGAA-30 . Each reaction contained 7 mL Lightcycler 480 SYBR GREEN I Master (containing Fast Start Taq polymerase, reaction buffer, dNTP mix, SYBR Green I dye and MgCl2), 0.5 mM of each, the sense and the antisense specic primers and 3 mL of the cDNA dilution in a nal volume of 10 mL. The amplication program consisted of a pre-incubation step for denaturation of the template cDNA (10 min, 95 C), followed by 45 cycles consisting of a denaturation step (10 s, 95 C), an annealing step (10 s, 60 C) and an extension step (10 s, 72 C). Aer each cycle, the uorescence was measured at 72 C (lex 470 nm and lem 530 nm). A negative control without cDNA templates was run in each assay. Real-time efficiencies were calculated from the given slopes in the LightCycler 480 soware using serial dilutions, showing all the investigated transcripts, high real-time PCR efficiency rates, and high linearity when different concentrations are
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used. PCR products were subjected to a melting curve analysis on the LightCycler and subsequently 2% agarose/TBE gel electrophoresis to conrm amplication specicity, Tm and amplicon size, respectively. Relative quantication aer PCR was performed by dividing the concentration of the target gene in each sample by the mean of the concentration of the three reference genes (housekeeping genes) in the same sample using the advanced relative quantication method provided by the LightCycler 480 analysis soware version 1.5 (Roche Diagnostics, Mannheim, Germany). Statistical analysis Cytotoxicity and gene expression data were presented as mean values standard error of the mean (SEM). Differences between groups were assessed by the Mann–Whitney-test. The SPSS®
SEM images and phase illustration of electrospun PCL–PEO blends: mat I, pure PCL of 20%, mat XI, pure PEO of 6%, indicated as blue and orange, respectively; mat II, PCL/PEO ¼ 18% : 0.6%, mat III, PCL/PEO ¼ 16% : 1.2%, mat IV, PCL/PEO ¼ 14% : 1.8%, “minor PEO in PCL” phase; mat V, PCL/PEO ¼ 12% : 2.4%, mat VI, PCL/PEO ¼ 10% : 3%, mat VII, PCL/PEO ¼ 8% : 3.6%, “co-continuous” phase of PEO and PCL; mat VIII, PCL/PEO ¼ 6% : 4.2%, mat XI, PCL/PEO ¼ 4% : 4.8%, and mat X, PCL/PEO ¼ 2% : 5.4, “minor PCL in PEO” phase. The composition of each mat and their phase illustration is summarized in the plot. Fig. 1
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program for Windows (Chicago, IL), version 17.0 was used. Results were considered statistically signicant at the p-values
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Ultraporous interweaving electrospun microfibers from PCL–PEO binary blends and their inflammatory responses† a b a Yan-Fang Li,‡ab Marina Rubert,‡a Hu ¨ snu ¨ Aslan, Ying Yu, Kenneth A. Howard, a a a Mingdong Dong, Flemming Besenbacher and Menglin Chen*
Production of one dimensional nanomaterials with secondary morphology exhibiting unique functions is challenging. Here we report for the first time that a nanoscale immiscible polymer blend solution electrojet can assemble into ultraporous interweaving microfibers. This intriguingly novel morphology originated from a blend of polycaprolactone (PCL) and polyethylene oxide (PEO) in a DCM–DMF mixed solution when the ratio between each component reached a threshold and when the electrospinning parameters were delicately controlled. The morphology, crystallinity, surface chemistry and wettabilities were characterized to understand the mechanism of formation. The interplay of the two semi-crystalline polymers and the pair of solvents/non-solvents with the electrospinning processing parameters was found to be critical for the formation of the unique structure. Furthermore, the interesting combination Received 21st November 2013 Accepted 22nd December 2013
of biocompatible, biodegradable PCL with protein-resistant PEO motivated us to assess its inflammation
DOI: 10.1039/c3nr06197c
responses on the RAW 264.7 macrophage cell line. All fibers were found to be biocompatible with low inflammation potential upon incubation, while compared with pure PCL nanofibers; the unique
www.rsc.org/nanoscale
interweaving microfibers induced a slightly higher inflammatory reaction.
Introduction Production of one dimensional nanomaterials with secondary morphology exhibiting unique functions is challenging. Electrospinning, one of the most promising nanotechnology innovations, has attracted great attention as a versatile method to produce continuous sub-micrometer-scale bers with large surface area, high porosity, controllable mechanical properties, and ease of functionalization1–4 for a wide range of applications in tissue engineering, drug delivery, ltration, sensors, and so on.5,6 While conventional electrospun bers usually have smooth surfaces without interior structures or specialized architecture, it is possible to develop bers with special structures, such as core–shell,7,8 hollow,9 and porous10–12 that may install material-specic functions for potential applications.13,14 The porosity within electrospun bers, both the surface and interior porosity, due to their increased surface area, is of a
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark. E-mail: [email protected]
b
Institute of Nanoscience and Nanotechnology, Central China Normal University, Wuhan 430079, China
† Electronic supplementary information (ESI) available: High resolution SEM image, AFM image, and TEM image of mat VI, PCL–PEO ¼ 10% : 3%. Morphology transformations of mat V: PCL–PEO ¼ 12% : 2.4% by changing different electrospinning parameters and solvent ratios. Contact angles of PCL–PEO bers before and aer water treatment. See DOI: 10.1039/c3nr06197c ‡ Yan-Fang Li and Marina Rubert contributed equally to this work.
3392 | Nanoscale, 2014, 6, 3392–3402
signicant importance in inuencing the nal application.15 A straightforward approach is using porogens. Poly-L-lactide (PLA) bers with interior porous structures were produced from electrospinning a PLA/PVP (polyvinylpyrrolidone) blend followed by selective removal of PVP in water.16 Interestingly, a delicate choice of polymers and solvents could allow phase separation into polymer rich and poor regions, with the latter becoming the pores. Porous electrospun PLA bers were reported by Bognitzki et al., where the porous structure was induced by the rapid evaporation of the solvent dichloromethane (DCM).10 Megelski et al.11 also investigated electrospun bers with different polymers/solvents with micro- and nanopores on the surface. Much attention has been focused on the development and investigation of binary polymer blends that allow the combination of desirable properties of different polymers with advantages over the synthesized novel polymeric materials.17,18 Many polymers are immiscible from the thermodynamic point of view, because the entropy contribution to Gibbs energy of mixing is negligible. Immiscible blends are particularly intriguing as they oen provide unique properties with special polymer architecture.19–21 Most of the investigated blended systems represent amorphous polymers; only few containing crystalline polymers have been studied, which were found to be complicated as both components are able to crystallize and provide various conned conditions for dening the crystallization behavior and resulting
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morphology.22 Crystallization is a solid–liquid separation process, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. It oen occurs during cooling or solvent evaporation; however crystallization during electrospinning has been scarcely investigated.23 Polycaprolactone (PCL) and polyethylene oxide (PEO) are both semicrystalline and they are immiscible. While the mixing of immiscible components is challenging,24 copolymers consisting of both PCL and PEO segment blocks exhibit a highly asymmetric lamellar structure and spherical or cylindrical structures.25–27 Here, a novel multi-lamellar cylindrical structure was fabricated via electrospinning of PCL–PEO blends directly from their homogeneous solutions in DCM/DMF. The morphology, crystallinity, surface chemistry and wettability of these bers were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD), X-ray photoelectron spectrometry (XPS), and contact angle (CA) measurements. Based on the morphology and structure characterization, the formation mechanism of the novel structure could be proposed. Both PCL and PEO are biocompatible and biodegradable and, thus, the most extensively studied polymers for electrospinning and widely used in tissue engineering28 and drug delivery.29–34 Since PEO is also well known to suppress protein adhesion35 and applied as antiinammatory polymeric coatings for implantable biomaterials and devices,36 the bers with different morphologies were applied on macrophage cell cultures. The toxicity of the different PCL–PEO bers as potential biomaterials was evaluated by quantication of the lactate dehydrogenase (LDH) activity, and the expression of inammation related marker genes was evaluated by real time RT-PCR. The cell attachment and morphology were further assessed by SEM and confocal microscopy.
Materials and methods
DMF (3 : 2) at room temperature and stirring until homogeneous solutions formed. The detailed information on the electrospinning solutions is listed in Table 1. The viscosity of the polymer solution was measured using a rheometer with a cone-plate conguration (Physica MCR 501, Anton Paar). The measuring cone was a CP50-1 with a diameter of 50 mm and an angle of 1 . Temperature of the cone and plate were kept at 20.0 C for all the solutions. The conductivity measurement was conducted using a laboratory conductivity meter (inoLab Cond 720), and the temperature of measuring was 24.1 C. The homogeneous polymer solutions were placed in a 1 mL syringe tted with a metallic needle of 0.9 mm inner diameter. The syringe was xed horizontally on the syringe pump (Model: KDS 101, KD Scientic), an electrode of high voltage power supply (Spellman High Voltage Electronics Corporation, MP Series) was clamped to the metal needle tip, and a grounded stationary rectangular metal collector (15 cm 20 cm) covered by a piece of clean aluminum foil was used for the ber collection. The electrospinning process was carried out under the following conditions: applied voltage ¼ 18 kV, feeding rate ¼ 1 mL h1, and distance between the tip of the needle and collector ¼ 12 cm. The experiments were carried out at room temperature, and the relative humidity was between 30% and 60%. The obtained bers were dried under vacuum (labconco Freezone Triad™) overnight to remove the excess solvents before further use. Scanning electron microscopy The morphologies of the electrospun bers were examined with a high-resolution scanning electron microscope (SEM) (FEI, Nova 600 NanoSEM). The bers were placed directly into the SEM chamber without any metal sputtering or coating; all the images were captured using a secondary electron detector with an acceleration voltage of 5 kV. Atomic force microscopy
Electrospinning The polymer solutions were prepared by dissolving PCL (Mw ¼ 70 000–90 000, Aldrich) and PEO (Mw ¼ 900 000, Aldrich) in DCM/
Aer immobilizing electrospun bers on at substrates the quantitative nanomechanical properties of the samples were characterized using a Dimension FastScan AFM (Bruker, Santa
Table 1 Electrospinning solution concentrations, viscosity, conductivity and the resulted electrospun fiber morphology, surface chemistry and wettability
% PEO Mat
%PCL (w/v)
%PEO (w/v)
Viscosity of solution (Pa s)
Conductivity of solution (ms cm1)
I II III IV V VI VII VIII IX X XI
20 18 16 14 12 10 8 6 4 2 0
0 0.6 1.2 1.8 2.4 3 3.6 4.2 4.8 5.4 6
1.06 1.01 0.79 0.67 1.33 1.98 3.12 6.60 3.35 3.78 3.81
1.9 2.0 2.2 1.8 2.5 2.6 2.9 3.1 3.1 3.5 2.7
a
Morphology
Solution
Surfacea
Contact angle ( )
Nanobers (0.92 0.61 mm) Pores on bers (3.01–3.39 mm)
0 8.08 16.51 25.32 34.52 44.16 54.26 64.86 75.98 87.68 100
0 33.28 39.29 51.18 45.83 58.14 65.65 58.47 64.01 82.67 100
117.9 89.1 84.6 28.8 19.9 23.0 33.1 37.0 34.7 40.3 16.4
Ultraporous interweaving microbers (3.45–4.78 mm) Melded bers Nanobers (0.56 0.11 mm)
% PEO ¼ ½ O–C (PEO)/(½ O–C (PEO) + O–C (PCL)).
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Barbara, CA, USA) under ambient conditions (temperature: 20 C and humidity: 51%). Peak force tapping was performed using triangular silicon nitride probes (nominal resonance frequency: 400 kHz, nominal spring constant: 4 N m1, and tip radius: 5 nm; FastScan-B, Bruker, USA). Topography, stiffness and adhesion images were acquired simultaneously. The linear scanning rate was set to 0.1–0.5 Hz with a scan resolution of 512 per line. The imaging force and other scan parameters were optimized to achieve the highest possible resolution also to avoid damaging the sample or wearing the tip. Collected images were further processed using SPIP soware (Image Metrology ApS, Lyngby, Denmark). Alignment of bers was quantitatively determined using Fast Fourier Transformation (FFT) calculations. Transmission electron microscopy The interior structures of the bers were detected by transmission electron microscopy (TEM) (Tecnai G2 F20 U-TWIN) at 200 kV. All the samples were prepared by electrospinning bers directly on carbon coated copper grids. X-ray diffraction X-ray diffraction measurements were carried out using a D8 Discover X-ray diffractometer with Cu Ka radiation (l ¼ 0.15406 nm) at a scan rate of 0.01 2q s1 for the characterization of the crystal structure of the electrospun bers. X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) was performed using a Kratos Axis UltraDLD instrument equipped with a monochromated Al KR X-ray source (hn ¼ 1486.6 eV) operating at 15 kV and 15 mA (225 W). A hybrid lens mode was employed during analysis (electrostatic and magnetic), with an analysis area of approximately 300 mm 700 mm. For each sample, a takeoff angle (TOA) of 0 (with respect to the sample surface) was used, allowing maximum probe depth (10 nm). Wide energy survey scans (WESSs) were obtained over the range of 0–1200 eV binding energy (BE) at a pass energy of 160 eV and used to determine the surface elemental composition. Contact angle The contact angle (CA) was measured by means of a Kr¨ uss Drop Shape Analysis System DSA100. A water droplet of 3 mL was dropped on the surface of the scaffold each time, and the CA values were recorded by continuous shooting mode with an interval of 5 ps1 for 15 s. Each sample was measured three times at different positions. CA values of bers aer water treatment were also measured in the same way. Before measuring the CA, bers were rstly immersed into Milli-Q water (18.2 MU cm at 25 C) for 48 h, rinsed with a large amount of Milli-Q water and then dried in a freeze drier overnight. Cell culture of RAW 264.7 cells on PCL and PEO–PCL bers The murine RAW 264.7 macrophage-like cell lines (ATCC, Manassas, USA) were routinely cultured in DMEM–glutamax
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(4.5 g L-D-glucose, () pyruvate) (GIBCO, Grand Island, NY) supplemented with 10% FBS (Biowhittaker, Walkersvile, MD) and antibiotics (100 IU and 100 IU streptomycin antibiotics) (Gibco, Grand Island, NY) at 37 C in a humidied atmosphere of 5% CO2. Cells were routinely subcultured in a 1 : 10 ratio before reaching conuence by scrapping. All experiments were performed aer 17 passages of the RAW 264.7 cells. Before seeding, three groups of bers (PCL, 1.8% PEO–14% PCL, and 3% PEO–10% PCL) of 12 mm Ø (1.1 cm2) were punched out and placed on the bottom of a sterile standard 48well plate, with a PCL ring (11 mm Ø) on the top. PCL rings were also placed into the TCP wells, which served as positive and negative reference groups. Raw 264.7 cells were seeded onto each ber at a density of 1.6 105 cells cm2 and maintained in DMEM–glutamax supplemented with 10% FBS and 1% antibiotics. In parallel, cells seeded on a 48-well TCP plate (() TCP) and TCP plus 0.1 mg mL1 lipopolysaccharide ((+) LPS TCP) (LPS, E. coli 055:B5, Schnelldorf, Germany) served as a negative and positive control, respectively. Cells were maintained under standard cell culture conditions (37 C in a humidied atmosphere of 5% CO2) for 24 hours. Aer 24 hours of cell seeding, the culture medium was collected to evaluate cytotoxicity (LDH activity). Cell attachment and morphology onto the bers was also visualized by SEM and confocal microscopy. In parallel, the expression of marker genes related to inammation was assessed by real-time RTPCR. To ensure that the cell characterization was done only on the cells growing onto the bers, samples were moved to a tube prior to gene expression analysis. Cell adhesion and morphology on PCL and PCL–PEO bers The cell morphology aer 24 hours of seeding was visualized using a confocal microscope (CLSM 700 Zeiss, Jena, Germany) and using a scanning electron microscope (SEM) (FEI, Nova 600 NanoSEM). For confocal microscopy images, RAW 264.7 cells adhered to the bers were xed with 4% formaldehyde in PBS for 15 minutes. For staining, cells were permeabilized in 0.2% Triton in phosphate buffered saline (PBS) (Sigma, Schnelldorf, Germany). The cytoskeleton of the cells was stained using 50 mg mL1 phalloidin Atto 488 (Sigma Aldrich, Schnelldorf, Germany) and the nuclei with Prolong® Gold Antifade Reagent with DAPI (Invitrogen, Carlsbad, CA, USA). SEM imaging was performed aer cell xation with glutaraldehyde 4% in PBS for 2 h followed by a dehydration process with ethanol as previously described.37 Cell viability (lactate dehydrogenase (LDH) activity) Aer 24 hours of cell seeding, the LDH activity in the collected culture media was taken as an indicator of membrane leakage/ cell lysis. The activity of the cytosolic enzyme was estimated according to the manufacturer's kit instructions (Roche Diagnostics, Mannheim, Germany), by assessing the rate of oxidation of NADH at 490 nm in the presence of pyruvate. Aer removing the background in the absorbance of the culture media without cells, results from all the samples were presented
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relative to the LDH activity in the medium of cells treated cultured on tissue culture plastic (TCP) (low control, 0% of cell death) and of cells cultured on TCP treated with 1% Triton X-100 (high control, 100% cell death). The percentage of LDH activity was calculated using the equation:
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Cytotoxicity (%) ¼ (exp. value low control)/ (high control low control) 100.
Total RNA isolation and gene expression of inammation markers by real-time RT-PCR The effect of bers to induce an inammation response was further studied by quantication of relative mRNA levels of selected inammation related markers aer 24 hours of cell seeding. Total RNA was isolated from cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. Total RNA was quantied at 260 nm using a Nanodrop spectrophotometer (IMPLE AH Diagnostics, Helsinki, Finland). 0.4 mg RNA was reverse transcribed to cDNA at 37 C for 60 min using a high capacity RNAto-cDNA kit (Applied Biosystems, Foster City, CA), according to the protocol of the supplier. Aliquots of each cDNA were frozen (20 C) until the PCR reactions were carried out. Real-time PCR was performed in a Lightcycler 480® (Roche Diagnostics, Mannheim, Germany) using SYBR green detection. Real time RT-PCR was done for three reference genes (18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TBP) and 3 target genes (tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b) and nitric oxide synthase (iNOS)). The primer sequences were as follows: 18s rRNA-F: 50 -GTAACCCGTTGAACCCCATT-30 ; 18s rRNA-R: 50 -CCATCCAATCGGTAGTAGCG-30 ; GAPDH-F: 50 -ACCCAGAAGACTGTGGATGG-30 ; GAPDH-R: 50 -CACATTGGG-GGTAGGAACAC-30 ; TBPF: 50 -AGAGAGCCACGGACAACTG-30 ; TBP-R: 50 -ACTCTAGCA TATTTTCTTGCTGCT-30 ; TNFa-F: 50 -GTAGCCCACGTCGTAG CAAAC-30 ; TNFa-R: 50 -ATCGGCTGGCACCACTAGTT-30 ; ILb-F: 50 GCCACCTTTTGACAGTGATGA-30 ; ILb-R: 50 -GATGTGCTGCTGC GAGATTT-30 ; iNOS-R: 50 -GCCACCAACAATGGCAACAT-30 ; iNOSF: 50 -TCGATGCACAACTGGGTGAA-30 . Each reaction contained 7 mL Lightcycler 480 SYBR GREEN I Master (containing Fast Start Taq polymerase, reaction buffer, dNTP mix, SYBR Green I dye and MgCl2), 0.5 mM of each, the sense and the antisense specic primers and 3 mL of the cDNA dilution in a nal volume of 10 mL. The amplication program consisted of a pre-incubation step for denaturation of the template cDNA (10 min, 95 C), followed by 45 cycles consisting of a denaturation step (10 s, 95 C), an annealing step (10 s, 60 C) and an extension step (10 s, 72 C). Aer each cycle, the uorescence was measured at 72 C (lex 470 nm and lem 530 nm). A negative control without cDNA templates was run in each assay. Real-time efficiencies were calculated from the given slopes in the LightCycler 480 soware using serial dilutions, showing all the investigated transcripts, high real-time PCR efficiency rates, and high linearity when different concentrations are
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used. PCR products were subjected to a melting curve analysis on the LightCycler and subsequently 2% agarose/TBE gel electrophoresis to conrm amplication specicity, Tm and amplicon size, respectively. Relative quantication aer PCR was performed by dividing the concentration of the target gene in each sample by the mean of the concentration of the three reference genes (housekeeping genes) in the same sample using the advanced relative quantication method provided by the LightCycler 480 analysis soware version 1.5 (Roche Diagnostics, Mannheim, Germany). Statistical analysis Cytotoxicity and gene expression data were presented as mean values standard error of the mean (SEM). Differences between groups were assessed by the Mann–Whitney-test. The SPSS®
SEM images and phase illustration of electrospun PCL–PEO blends: mat I, pure PCL of 20%, mat XI, pure PEO of 6%, indicated as blue and orange, respectively; mat II, PCL/PEO ¼ 18% : 0.6%, mat III, PCL/PEO ¼ 16% : 1.2%, mat IV, PCL/PEO ¼ 14% : 1.8%, “minor PEO in PCL” phase; mat V, PCL/PEO ¼ 12% : 2.4%, mat VI, PCL/PEO ¼ 10% : 3%, mat VII, PCL/PEO ¼ 8% : 3.6%, “co-continuous” phase of PEO and PCL; mat VIII, PCL/PEO ¼ 6% : 4.2%, mat XI, PCL/PEO ¼ 4% : 4.8%, and mat X, PCL/PEO ¼ 2% : 5.4, “minor PCL in PEO” phase. The composition of each mat and their phase illustration is summarized in the plot. Fig. 1
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program for Windows (Chicago, IL), version 17.0 was used. Results were considered statistically signicant at the p-values
