In this study, liquid plasma treatment was used to improve the morphology of Poly-CaproLactone (PCL) NanoFibers (NFs), followed by performing a Dielectric Barrier Discharge (DBD) plasma surface modification to enhance the hydrophilicity of electrospun mats generated from plasma-modified PCL solutions. Cell interaction studies performed after 1 day and 7 days clearly revealed the highly increased cellular interactions on the double plasma-treated nanofibers compared to the pristine ones due to the combination of (1) a better NF morphology and (2) an increased surface hydrophilicity

Asadian, Mahtab

De Geyter, Nathalie

Declercq, Heidi

Grande, Silvia

Morent, Rino

Nikiforov, Anton

English

Ghent University Academic Bibliography

Journal of Physics: Conference SeriesPAPER • OPEN ACCESSEffects of pre- and post-electrospinning plasmatreatments on electrospun PCL nanofibers toimprove cell interactionsTo cite this article: M Asadian et al 2017 J. Phys.: Conf. Ser. 841 012018 View the article online for updates and enhancements.Related contentSurface modified electrospun nanofibrousscaffolds for nerve tissue engineeringMolamma P Prabhakaran, J Venugopal,Casey K Chan et al.-Incorporation of ciprofloxacin/laponite inpolycaprolactone electrospun nanofibers:drug release and antibacterial studiesKaleemullah Kalwar, Xuan Zhang,Muhammad Aqeel Bhutto et al.-C Wan and B ChenChaoying Wan and Biqiong Chen-This content was downloaded from IP address 157.193.64.176 on 24/01/2018 at 16:111Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distributionof this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.Published under licence by IOP Publishing Ltd12345678907th Young Researcher Meeting  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 841 (2017) 012018  doi :10.1088/1742-6596/841/1/012018Effects of pre- and post-electrospinning plasmatreatments on electrospun PCL nanofibers toimprove cell interactionsM Asadian*1, S Grande*1, R Morent1, A Nikiforov1, H Declercq2, NDe Geyter11 Faculty of Engineering & Architecture, Department of Applied Physics, Research UnitPlasma Technology (RUPT), Ghent University, Sint-Pietersnieuwstraat 41 B4, 9000 Ghent,Belgium2 Faculty of Medicine and Health Science, Department of Basic Medical Sciences, TissueEngineering and Biomaterials Group, Ghent University, De Pintelaan 185 6B3, 9000 Ghent,BelgiumE-mail: Mahtab.Asadian@UGent.be, Silvia.Grande@UGent.beAbstract. In this study, liquid plasma treatment was used to improve the morphology of Poly-ǫ-CaproLactone (PCL) NanoFibers (NFs), followed by performing a Dielectric Barrier Discharge(DBD) plasma surface modification to enhance the hydrophilicity of electrospun mats generatedfrom plasma-modified PCL solutions. Cell interaction studies performed after 1 day and 7days clearly revealed the highly increased cellular interactions on the double plasma-treatednanofibers compared to the pristine ones due to the combination of (1) a better NF morphologyand (2) an increased surface hydrophilicity.1. IntroductionThe application of electrospun nanofibers (NFs) in tissue engineering is increasing owing to theirmorphological similarity to the ExtraCellular Matrix (ECM) as most of the ECM proteins havea fibrous structure with diameters in the nanometer or sub-micrometer range [1]. Nevertheless,a great challenge in electrospinning is the preparation of a suitable polymer solution because thesystem requires a balance of forces controlled by rheological behavior, conductivity and surfacetension. In many cases, researchers have used mixed solvents or even additives to make solu-tions compatible to the electrospinning process [2]. However, within this work, a non-thermalAtmospheric Pressure Plasma Jet (APPJ) directly generated in a polymer solution will be ex-plored trying to improve the electrospinnability and final NF quality. The applied APPJ set-upallows a close, intense contact between the plasma and the polymer solution which can lead toa very efficient effect on the quality of the resultant fibers [3 - 5]. As PCL is a good polymericcandidate for tissue engineering applications due to its numerous advantages such as good me-chanical properties (highly elastic), chemical stability, excellent biocompatibility, research willbe focused on PCL in this study. Nonetheless, due to its highly hydrophobic structure, PCLshows very poor cellular interactions. To cope with this problem, a Dielectric Barrier Discharge(DBD) plasma surface treatment will also be performed on selected nanofibrous samples after212345678907th Young Researcher Meeting  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 841 (2017) 012018  doi :10.1088/1742-6596/841/1/012018the electrospinning process [6, 7]. For this purpose, PCL-NFs will be exposed to an argonplasma in an effort to improve their hydrophilicity as this is known to positively affect cellularinteractions [8]. Plasma surface modification is a very attractive technique for this purpose asit does not significantly change the scaffold morphology nor its mechanical properties [9, 10].In this study, the PCL solution viscosity and conductivity will be monitored before and afterAPPJ treatment, while Scanning Electron microscopy (SEM) will be used to evaluate the mor-phology of the resultant NFs. Furthermore, PCL-NFs will also be seeded with HFFs (Humanforeskin fibroblasts) to perform cell adhesion tests after 1 and 7 days. For this purpose, 4 dif-ferent samples will be examined: (1) untreated scaffolds, (2) scaffolds plasma-treated beforethe electrospinning process, (3) scaffolds plasma-treated after the electrospinning process and(4) scaffolds plasma-treated before and after the electrospinning step. Cell interactions on thedifferent samples will be assessed by fluorescence microscopy and SEM.Figure 1. A schematic of the APPJ (left) to treat the PCL solution and the DBD (right) toimprove the hydrophilicity of the PCL nanofibers.2. ExperimentalPCL with a MW of 80000 g/mol, chloroform and N,N-Dimethylformamide were purchased fromSigma-Aldrich and used without further purification. A solution of 5% of PCL in CHCl3+DMF(ratio of 9:1 (w/w)) was prepared at room temperature by stirring for 2 hours. Half of theobtained polymer solution was electrospun without any plasma pre-treatment step, while theother half of the polymer solutions was treated with the atmospheric pressure plasma jet (APPJ)schematically presented in Fig. 1 for 5 minutes making use of an argon flow rate of 0.9 slm(standard liters per minute) and an applied voltage of 7.5 kV. Before and after the APPJmodification step, the solution was characterized by measuring its conductivity and viscositymaking use of a Mettler Toledo FiveEasy conductivity meter equipped with an InLab 720 probeand a Brookfield DV2T extra viscometer, respectively. After performing the APPJ modificationstep, untreated and plasma-modified PCL solutions were immediately electrospun making use312345678907th Young Researcher Meeting  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 841 (2017) 012018  doi :10.1088/1742-6596/841/1/012018of a Nanospinner 24 electrospinning machine manufactured by Inovenso (Turkey). This machineconsists of a copper tip which is fed with the PCL polymer solution at a rate of 3.5 ml/h. Ahigh voltage of 25 kV is applied to this tip, while a grounded rotating drum (rotation speed: 100rpm) placed at 20 cm from the tip acts as collector plate. Round cover glasses with a diameterof 12 mm were fixed to the rotating drum and used as collecting substrates.In a next step, the obtained PCL nanofibers were exposed to a DBD plasma schematicallypresented in Fig. 1(right) to increase their hydrophilicity. The DBD treatments were performedin argon at a discharge power of 1.5 W at medium pressure (50 kPa). A treatment time of 30seconds was selected since this treatment duration was found to result in the highest possiblehydrophilicity combined with negligible changes in NF morphology. The morphology of thePCL-NFs before and after APPJ liquid treatment and DBD plasma surface treatment werecharacterized with a scanning electron microscope (SEM; JSM-6010 PLUS/LV; JEOL). SEMimages are acquired with an accelerating voltage of 7 kV, after coating the samples with a thinlayer of gold making use of a sputter coater (JFC-1300 autofine coater, JEOL). The averagediameter of nanofibers is calculated over 20 fibers using ImageJ (National Institutes of Health,USA) analysis software.In a final step, HFF cells were cultured on untreated, pre-electrospinning plasma treated, post-electrospinning plasma treated and pre- and post-electrospinning plasma treated NFs afterexposure to UV light for 30 minutes as a sterilization step. For this purpose, a density of 40.000cells/500 µl of culture medium per scaffold was used. The cell seeded scaffolds were incubatedat 37◦C under 5% CO2 atmosphere for 1 and 7 days and Tissue Culture PolyStyrene (TCPS)was used as positive control. Live/dead cell staining was applied to evaluate cell viability:the cell seeded scaffolds were incubated in 2 µl (1 mg/ml) of calcein-AM and 2 µl (1mg/ml)propidium iodide in 1 ml phosphate buffered saline (PBS), after which they were imaged witha fluorescence microscope (Olympus; IX 81) with appropriate filters [8]. The morphology ofthe HFF cells on the scaffolds after 1 and 7 days was also evaluated by SEM after performinga cell fixation and dehydration step. The preparation procedure included cell washing withPBS followed by fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 hour. Thecells were subsequently dehydrated by immersion through increasing ethanol series (50%, 70%,85%, 95% and 100%) for 10 min. Afterwards, samples were immersed in hexamethyldisilazane(HDMS) for 10 min.3. Results and discussion3.1. APPJ treatment of the PCL polymer solution: solution properties and PCL-NFmorphologyThe conductivity and viscosity of the PCL polymer solution before and after APPJ treatmentcan be found in Table 1: from reported values it is evident that plasma exposure can stronglyincrease these parameters. SEM images of electrospun PCL-NFs generated from untreated andplasma treated PCL solutions are also displayed in Fig. 2. This figure clearly shows that thenanofibrous scaffold obtained from the untreated polymer solution presents a high density ofbeads (on average 2000 beads per mm2) and an average fiber diameter of approximately 300 nm(high standard deviation due to the presence of beads). After 5 minutes of treatment with theplasma jet, the beads however completely disappear resulting in a network of elongated, uniformfibers with an average fiber diameter of approximately 500 nm.412345678907th Young Researcher Meeting  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 841 (2017) 012018  doi :10.1088/1742-6596/841/1/012018Table 1. Polymer solution characteristics before and after APPJ treatmentUntreated APPJ for 5 minConductivity (µS)/cm 0.25± 0.05 5.1± 0.1Viscosity (cP) 85± 5 320± 5Fibers diameter (nm) 300± 150 500± 50Figure 2. SEM images of nanofibers generated from A) an untreated PCL solution and B) aPCL solution treated with an APPJ for 5 minutes.As mentioned before, plasma treatment is a very useful method to modify the chemicalproperties of a polymer surface. Plasma incorporated polar chemical groups are known toincrease the surface energy of polymers and thus increase the surface wettability. The untreatednanofibers have a contact angle of approximately 127◦ which decrease to values of 55◦, 64◦and 95◦ for the nanofibers plasma treated with combined plasma treatments, DBD treated andAPPJ treated respectively.3.2. Live/dead immune microscopy and cell morphology using SEMThe cellular interactions on differently prepared PCL scaffolds have also been investigated 1day and 7 days after cell seeding and the images obtained by SEM and fluorescent microscopyare presented in Figs. 3 and 4 respectively. These images clearly demonstrate the differencesin cellular interactions between untreated, APPJ treated, DBD treated and combined plasmatreatments on PCL-NFs. On untreated samples, after 1 day of cell seeding, mostly round andthus poorly attached cells can be observed. In addition, after 7 days of cell seeding, most of thepresent cells still show a round morphology while also quite a few cells appear to be dead (redcolor in Fig. 4). Cellular interactions on untreated samples are thus very poor. On sampleswhich have been treated with the DBD, one can see that after 1 day and 7 days of cell seeding,more live cells are present on the samples and the cells adhere better to the sample surface, whichcan be evidenced from their spread morphology. These better cellular interactions comparedto the untreated sample is most likely due to the enhanced hydrophilicity, which is known topositively affect cell-material interactions. When looking at the sample which has been treatedwith the APPJ alone, one can see that also in this case, cells nicely adhere to the sample asthey present an elongated morphology, which is most likely due to the presence of uniform fibersinstead of beads. However, the best cell-material interactions can be observed on the combined512345678907th Young Researcher Meeting  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 841 (2017) 012018  doi :10.1088/1742-6596/841/1/012018APPJ and DBD plasma-treated samples: in this case, a high number of nicely elongated cellscan be observed on the surface, even after only 1 day after cell seeding. After 7 days, a layer ofcells is almost completely covering the nanofibers (as can be nicely observed in Fig. 3), whichwas not at all the case for the other investigated samples. These enhanced cellular interactionscompared to the APPJ treated samples can again be related to the enhanced hydrophilicityupon DBD treatment. One can therefore conclude that a double plasma treatment is able tostrongly enhance the cellular interactions on PCL nanofibers.Figure 3. SEM images. First row: after 1 day, A) untreated samples, B) fibers with a DBDplasma treatment, C) APPJ plasma treatment, D) APPJ+DBD plasma treatment. Second row:after 7 days of seeding, E) untreated, F) DBD treatment, G) APPJ treatment, H) APPJ+DBDplasma treatment.Figure 4. Fluorescence microscopy images. First row: after 1 day, A) untreated samples,B) fibers with a DBD plasma treatment, C) APPJ plasma treatment, D) APPJ+DBD plasmatreatment. Second row: after 7 days of seeding, E) untreated, F) DBD treatment, G) APPJtreatment, H) APPJ+DBD plasma treatment.612345678907th Young Researcher Meeting  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 841 (2017) 012018  doi :10.1088/1742-6596/841/1/0120184. ConclusionAfter electrospinning an untreated PCL solution, the generated NFs presented a large amountof beads. It was however found that in the liquid plasma-treated fiber mesh, beads completelydisappeared leading to more uniform fibers in diameter and in morphology. However, the resul-tant, elongated NFs still showed to have a strong hydrophobic behavior which is unwanted inthe foreseen biomedical applications. DBD plasma treatment in argon of the NFs is thereforealso performed to significantly improve their hydrophilicity. Cell studies revealed that HFFscultured on PCL-NFs present a considerably higher cell adhesion and proliferation on combinedAPPJ and DBD plasma-treated NFs compared to pristine NFs. These results suggest that dou-ble plasma-modified NFs may have a large potential for the efficient replacement of damagedtissues in the future.AcknowledgmentsThe research leading to these results has received funding from the European ResearchCouncil under the European Unions Seventh Framework Program (FP/2007-2013): ERC GrantAgreement number 335929 (PLASMATS).References[1] Teo W E, Ramakrishna S 2006, Nanotechnology, 17 (14), R89[2] MacDiarmid A G et al. 2001, Synthetic Metals, 119, 27-30[3] Shi Q et al. 2011, Journal of Polymer Science Part B: Polymer Physics, 49 (2), 115-122[4] Hsu C M, Shivkumar S 2004, Macromolecular Materials and Engineering, 289 (4), 334-340[5] Colombo V, Fabiani D, Focarete M L, Gherardi M, Gualandi C, Laurita R and Zaccaria M 2014, PlasmaProcesses and Polymers, 11 (3), 247-255[6] Desmet T, Morent R, De Geyter N, Leys C, Schacht E, Dubruel P 2009, Biomacromolecules, 10 (9), 2351-2378[7] Jacobs T, Morent R, De Geyter N, Dubruel P, Leys C 2012, Plasma Chemistry and Plasma Processing, 32(5), 1039-1073[8] Yildirim E, Ayan H, Vasilets V, Fridman A, Guceri S, Sun W 2008, Plasma Processes and Polymers, 5 (1),58-66[9] Morent R, De Geyter N, Verschuren J, De Clerck K, Kiekens P, Leys C 2008, Surface and Coatings Technology,202 (14), 3427-3449[10] Morent R, De Geyter N, Leys C, Gengembre L, Payen E 2007, Textile Research Journal, 77 (7), 471-488

Effects of pre- and post-electrospinning plasma treatments on electrospun PCL nanofibers to improve cell interactions

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Effects of pre- and post-electrospinning plasma treatments on electrospun PCL nanofibers to improve cell interactions

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