Asadian, Mahtab

Cornelissen, Maria

De Geyter, Nathalie

Declercq, Heidi

Morent, Rino

Ghent University Academic Bibliography

XXXI International Conference on Surface Modification Technologies (SMT31) JULY 5-7, 2017, MONS, BELGIUM  _____________________________ * Corresponding author. Tel.: +32-9-264-3837; fax: +32-9-264-4198. E-mail address: nathalie.degeyter@ugent.be    EFFECTS OF PLASMA TREATMENT ON THE SURFACE CHEMISTRY, WETTABILITY, AND CELLULAR INTERACTIONS OF NANOFIBROUS SCAFFOLDS  M. Asadiana, H. Declercqb, M. Cornelissenb, R. Morenta, N. De Geytera,* a Research Unit Plasma Technology (RUPT), Department of Applied Physics, Faculty of Engineering & Architecture, Ghent University, Sint-Pietersnieuwstraat 41, 9000 Ghent, Belgium b Tissue Engineering Group, Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, 6B3, 9000 Ghent, Belgium  _________________________________________________________________________________________________________________________________   Abstract  In this study, poly-ε-caprolactone (PCL) nanofibrous mats (Ø: 107 ± 26 nm) were prepared by electrospinning and subsequently subjected to a dielectric barrier discharge (DBD) plasma treatment sustained in three different gases (argon, ammonia/helium and nitrogen). In a next step, water contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to characterize the surface properties of the PCL nanofibers before and after plasma treatment. Cell behavior on the fibers was also evaluated in detail by live/dead fluorescence microscopy performed one day and seven days after seeding of human foreskin fibroblasts (HFFs). In addition, the cells morphology on the nanofibers was observed with SEM. The obtained results revealed that the hydrophilicity of PCL nanofibers can be significantly increased due to the incorporation of nitrogen-containing groups after plasma modifications in NH3/He and N2 and due to the incorporation of oxygen-containing groups after plasma modification in argon. In addition, these increases in hydrophilicity occur without inducing any morphological changes to the PCL nanofibers. Cell studies also reveal that cell adhesion and proliferation on the nanofibers were strongly increased due to the performed plasma treatments, most likely as a result of the increased surface hydrophilicity. Plasma surface modification is thus a very promising strategy to modify the surface of nanofibrous materials to be used in tissue engineering applications.   Keywords: nanofibers, plasma treatment, cell interactions  _________________________________________________________________________________________________________________________________ 1. Introduction The application of nanofibers (NFs) in tissue engineering has been strongly increasing over the past years due to their morphological similarity to the extracellular matrix (e.g. to restore damaged tissues such as cartilage, tendon, skin and nerve) (Pham et.al., 2006). The surface properties (i.e. wettability, surface chemistry) of these engineered nanofibrous scaffolds play an important role in being successful in tissue engineering applications as they strongly affect cell–extracellular matrix interactions (Mrksich, 2000). Unfortunately, most of the biodegradable polymers suitable for nanofibrous tissue engineering scaffolds (PCL, polylactic acid (PLA),…) have a highly hydrophobic nature resulting in poor cell-material interactions (Kweon et al., 2003) . Within this work, we aim to alter the surface chemistry of PCL nanofibers by incorporating various functional groups during a non-thermal plasma treatment in an effort to positively affect the interactions between cells and PCL nanofibers.    2. Materials and methods A mixed solvent of formic acid and acetic acid (Sigma-Aldrich) at a ratio of 9:1 was used to dissolve PCL (Sigma-Aldrich) with a MW of 80000 g/mol to make a PCL solution of 14% (w/v). The obtained solution was introduced to an electrospinning machine (Nanospinner24, Inovenso, Turkey) making use of the following conditions: an applied voltage of 33 kV, distance between needle and collector equal to 15 cm and the solution flow rate set to 0.7 ml/h at room temperature. Next, the obtained NFs were modified using a DBD plasma. The discharge was generated between two round electrodes: the upper electrode is a stainless steel mesh (Ø: 50 mm), while the lower electrode is a copper plate (Ø: 50 mm) covered with Al2O3. The gap between both electrodes was maintained at 1 mm and the mesh electrode was connected to an AC high voltage source with a frequency of 50 kHz while the lower electrode was grounded through a capacitor of 10 nF. After the nanofibrous materials to be treated were placed on the lower electrode, the plasma reactor was pumped down to 0.2 mbar and then filled with the discharge gas for 3 minutes at a rate of 3 standard liters per minute (slm) to reach a pressure of  900 mbar. Afterwards, the gas flow rate was decreased to 1 slm and the pressure in the plasma reactor was lowered to 50 mbar. The plasma treatments in this work were performed at 50 mbar in three different discharge gases (argon, nitrogen, and a 10% ammonia/90% helium mixture) at a discharge power of 2.5, 6.0 and 12.0 W respectively, and with a fixed treatment time of 70 seconds.   The morphology of the PCL-NFs before and after DBD plasma treatment was characterized by scanning electron microscopy (SEM, JSM-6010 PLUS/LV, Jeol, USA) to detect possible changes in the morphology of the NFs and to measure the average fiber diameter with ImageJ software. The hydrophilicity of the nanofibrous scaffolds was also examined with a drop-shape analysis system (DSA25, Krüss GmbH, Germany) by using deionized water droplets (2 µl) at room temperature. The chemical composition of the polymer surfaces was determined by XPS, which was performed with a PHI Versaprobe II device, using a non-monochromatic Al Kα-beam (hν = 1486.6 eV) operating at 43.5 W. All measurements were conducted in a vacuum of at least 10−6 Pa and the photoelectrons were detected with a hemispherical analyzer positioned at an angle of 45° with respect to the normal sample surface. Survey scans were recorded with a pass energy of 187.85 eV and the elements present on the surface were identified from these scans. The samples were measured at six locations per sample and the present elements were quantified with Multipak software using a Shirley background by applying the relative sensitivity factors supplied by the manufacturer.   HFF cells were cultured on the treated and untreated NFs after exposure to UV light for 30 minutes (sterilization step) at a density of 40.000 cells/1000 µl of culture media per scaffold. The cell seeded scaffolds were incubated at 37°C under 5% CO2 for 1 and 7 days. Tissue culture polystyrene (TCPS) was used as a positive control. Live/dead cell staining was used to evaluate cell viability, the cell seeded scaffolds were incubated in 2 μl (1 mg/ml) of calcein-AM and 2 μl (1 mg/ml) propidium iodide in 1 ml phosphate buffer saline (PBS) and were subsequently imaged with a fluorescence microscope (Olympus, IX 81) with appropriate filters. The morphology of the HFF cells was also evaluated making use of SEM after performing a cell fixation step. 3. Results and Discussion In a first step, the electrospun PCL-NFs were examined making use of SEM to examine their morphology and the result is shown in Fig.1. This SEM image reveals a smooth PCL fiber morphology without the presence of beads and a randomly interconnected structure making use of previously optimized electrospinning parameters. The average fiber diameter was also determined and was found to be 107±2 nm. In a next step, the PCL-NFs were plasma treated in nitrogen, NH3/helium and argon. All these plasma treatments were found to strongly improve the wettability of the hydrophobic PCL nanofiber surfaces. The untreated nanofibers had a contact angle of approximately 135° which considerably decreased to a value of 35°, 24° and 13° for the nanofibers plasma treated in argon, nitrogen and NH3/helium, respectively. In addition, it was also observed from the SEM images of the plasma modified samples (an example after argon plasma treatment is shown in Fig.1) that the surface morphology of the nanofibers was not affected by the different plasma treatments.                         Fig.1. SEM images of PCL nanofibers (a) before and (b) after argon plasma treatment.  In a next step, the chemical composition of the untreated and plasma treated PCL NFs was examined making use of XPS. Based on XPS survey scans, the surface elemental chemical composition was determined before and after the different plasma treatments and the results are shown in Table 1.       Table 1. Elemental composition of the untreated and plasma modified PCL nanofibrous scaffolds Sample C (at%) O (at%) N (at%) Untreated 77.97 22.03 0 Argon plasma 76.78 23.22 0 Nitrogen plasma 66.50 16.72 16.78 NH3/He plasma 75.78 16.51 7.71  As can be seen in Table 1, argon plasma modification results in a small increase in oxygen content on the surface. A possible mechanism for this oxygen incorporation in an argon plasma is the plasma-induced partial decomposition of PCL molecular chains on the nanofiber surfaces. Indeed, plasma treatment is known to remove hydrogen atoms from polymer chains leaving free radicals on the polymeric surface. When the plasma-treated nanofibers are then subsequently exposed to air, oxygen or moisture can react with these radicals resulting in the formation of oxygen-containing groups on the PCL surface (Yan et al., 2013). Table 1 also clearly shows that an NH3/helium plasma treatment results in a significant nitrogen incorporation of approximately 7.7%, while plasma treatment in nitrogen leads to more than twice this nitrogen incorporation (16.8%). In both cases, the relative oxygen content decreases as additional nitrogen atoms are added to the surface, however, it is anticipated that no oxygen is removed from the PCL surfaces. Based on the XPS results, it can thus be concluded that argon plasma treatment results in the incorporation of polar oxygen functional groups, while in the case of NH3/helium and nitrogen plasma treatments nitrogen polar groups are created on the PCL-NF surfaces.    The cellular interactions on the differently prepared PCL scaffolds have also been investigated 1 day and  7 days after cell seeding and the images obtained by SEM and fluorescent microscopy are presented in Fig.2. These images clearly demonstrate strong differences in cellular interactions between the untreated sample on the one hand and the plasma treated samples on the other hand. On the untreated sample, 1 day after cell seeding, cells mostly cluster and thus poorly attach to the sample. In addition, 7 days after cell seeding, most of the cells present on the untreated sample still show clusters while also quite a few cells appear to be dead (red color in Fig.2). It can thus be concluded that cellular interactions on the untreated sample are thus very poor. On PCL samples which have been treated with the different DBDs, one can see that 1 day after cell seeding, more live cells are present on the samples while the cells also adhere better to the sample surface, which can be evidenced from their spread morphology. On all plasma treated samples, 7 days after cell seeding, a layer of cells is almost completely covering the nanofibers (as can be nicely observed in Fig.2). The better cellular interactions on the plasma modified samples compared to the untreated sample are most likely due to the enhanced surface hydrophilicity, which is known to positively affect cell-material interactions.   a b                   4. Conclusion In this work, non-thermal plasma treatments have been performed in different gases (argon, nitrogen and NH3/He) to modify the surface properties of electrospun PCL nanofibers. It was observed that all performed plasma treatments were able to significantly increase the hydrophilicity of the PCL nanofibers and this without causing any morphological changes to the nanofibers. These increases in hydrophilicity could be attributed to the incorporation of nitrogen-containing functional groups after nitrogen and NH3/helium plasma treatment and due to the incorporation of oxygen-containing functional groups after argon plasma treatment. The induced chemical changes were also found to positively affect the adhesion and proliferation behavior of HFFs on the PCL nanofibers. It was observed that more live cells were present on the plasma-modified samples and that the cells adhere better on the plasma-modified samples which was evidenced by their spread morphology. Plasma modifications thus strongly increase cell-materials interactions on PCL nanofibrous scaffolds most likely due to the enhanced hydrophilicity which is known to positively affect these interactions. These results thus suggest that plasma modified NFs may have a large potential to act as tissue engineering scaffolds in the near future.   Acknowledgements This research has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP2007-2013): ERC Grant Agreement number 335929 (PLASMATS) and ERC Grant Agreement number 279022 (PLASMAPOR).  References Pham, Q. P., Sharma, U., & Mikos, A. G. 2006. Electrospinning of polymeric nanofibers for tissue engineering applications: a review. Tissue engineering, 12(5): 1197-1211. Kweon, H., Yoo, M. K., Park, I. K., Kim, T. H., Lee, H. C., Lee, H.-S., Oh, J.-S., Akaike, T., & Cho, C.-S. 2003. A novel degradable polycaprolactone networks for tissue engineering. Biomaterials, 24(5): 801-808. Mrksich, M. 2000. A surface chemistry approach to studying cell adhesion. Chemical Society Reviews, 29(4): 267-273. Yan, D., Jones, J., Yuan, X., Xu, X., Sheng, J., Lee, J. M., Ma, G., & Yu, Q. 2013. Plasma treatment of electrospun PCL random nanofiber meshes (NFMs) for biological property improvement. Journal of Biomedical Materials Research Part A, 101(4): 963-972.  Fig. 1. Images of HFF cells at (a) day 1 and (b) day 7, cultured on untreated and different plasma treated samples obtained with fluorescence and scanning electron microscopy. 

Effects of plasma treatment on the surface chemistry, wettability, and cellular interactions of nanofibrous Scaffolds

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Effects of plasma treatment on the surface chemistry, wettability, and cellular interactions of nanofibrous Scaffolds

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