Cell Adhesion on Polycaprolactone Modified by Plasma Treatment

1 Jozef Stefan Institute, Jamova Cesta 39, 1000 Ljubljana, Slovenia 2Jozef Stefan International Postgraduate School, Jamova Cesta 39, 1000 Ljubljana, Slovenia 3National Institute of Biology, Večna Pot 111, 1000 Ljubljana, Slovenia 4International and Inter University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, Kerala 686560, India 5Department of Materials Science and Engineering, Technion-Israel Institute of Technology, De-Jur Building, Technion City, 3200003 Haifa, Israel 6School of Pure and Applied Physics, Mahatma Gandhi University, Kottayam, Kerala 686560, India 7School of Chemical Sciences, Mahatma Gandhi University, Kottayam, Kerala 686560, India


Introduction
Polymeric materials are nowadays frequently used in various medical applications including artificial implants, tissue engineering scaffolds, wound dressings, and drug delivery systems [1][2][3][4].Many of such applications usually require modification of surface properties of the polymer to improve its biocompatibility, cell adhesion and proliferation, and attachment of bioactive functional groups [5][6][7][8].Various methods like plasma treatment, grafting of polymer brushes, or applying different coatings can be used for polymer surface modification.
For the past few decades, nanofibers have gained much importance due to the potential applications in broad areas of technological applications.Many of the potential uses of nanofibrous membranes are related to high porosity, large surface area, and small pore distribution.There are many methods for the fabrication of nanofibrous membranes; however, the most successful method is the electrospinning.Electrospinning can produce continuous nanofibers from submicron diameter scale down to nanometer diameter scale through an electrically charged jet of polymer solution [9,10].
Electrospun three-dimensional polymeric scaffolds are becoming important in tissue engineering applications, especially in bone regeneration [11,12] and skin reconstruction [13,14].When cells attach to the scaffold, the scaffold has to offer optimal support and conditions for cell growth that leads to the formation of a new tissue.Tissue engineering scaffolds are often made of polymers like polycaprolactone (PCL) or polylactic acid (PLA) [15,16].Such polymeric scaffolds are biocompatible and biodegradable, which means that it is not necessary to remove them from the human body after implantation, because in vivo they slowly degrade to nontoxic products [17].However, such scaffolds require surface modification to improve cell adhesion and proliferation [18].Plasma treatment is one of the most useful techniques, because it allows for incorporation of different functional groups on the surface of the treated polymer.By proper choice of gas it is possible to manipulate these functional groups and change their nature, this way altering their surface wettability and surface energy, which both have a drastic impact on protein and cell adhesion [19][20][21].Some authors have used oxygen or air plasma for surface modification of PCL and found increased surface hydrophilicity, surface energy, surface roughness, and total amount of oxygen functional groups on PCL surface [22,23].They have also observed better attachment and proliferation of osteoblast cells [18,24,25].Wulf et al. have used ammonia plasma and found that modified surface did not affect the mouse fibroblast cell viability [26].Helium and argon plasma were used as well.Both plasmas led to incorporation of oxygen functional groups to PCL.In case of helium, the nitrogen functional groups were found on the surface of treated PCL as well.Unfortunately, the biological cell response to modified PCL was not tested [22].
Because different plasmas can influence cell adhesion differently and, furthermore, different cells can behave differently on the same surface [27], more systematic research of modified polymer surfaces is needed.In the present investigation we investigated the surface modifications of PCL polymer induced by oxygen (O 2 ), ammonia (NH 3 ), or sulphur dioxide (SO 2 ) plasma treatment.NH 3 plasma was chosen to introduce amino groups which are important in many biological processes.For SO 2 plasma there is a lack of scientific literature, although it is known that sulphate groups can play an important role in antithrombogenicity of the surface [28,29], whereas O 2 plasma was used as a control, because it is usually very efficient for achieving good endothelialization [27].Therefore, we have tested the effect of these modifications on cell adhesion and proliferation of human umbilical vein endothelial cells (HUVEC).

Fabrication of Electrospun PCL Scaffolds.
Electrospinning was carried out using PCL solutions with a polymer concentration of 15 wt.% which were prepared in acetone.The clear solution without any turbidity was taken in a 15 mL plastic syringe with 20-gauge blunt tip needle and electrospun at a high direct current (DC) voltage of 18 kV, a flow rate of 1 mL/h, and a tip to collector distance of 15 cm.A thin aluminum sheet with an approximate dimension of 10 cm × 7 cm was used as the collector.Upon applying a high DC voltage using a high-voltage power supply, a fluid jet was ejaculated from the tip of the needle.As the jet accelerated toward the target, the solvent evaporated and polymer nanofibers get deposited on the aluminum sheet as a fibrous membrane.All the electrospinning process was carried out at a temperature of 28 ∘ C and a relative humidity of 60%.
Scanning electron microscopic (SEM) images of the fabricated electrospun PCL membranes are shown in Figure 1.

Plasma
Treatment.PCL samples were treated in a discharge tube made from Pyrex glass, 80 cm long and 4 cm in diameter.The tube was pumped with a rotary pump operating at a nominal pumping speed of 80 m 3 h −1 .A coil of 6 turns was mounted in the centre of the tube.Plasma was created by an RF generator coupled to the coil via a matching network.The generator operated at the standard frequency of 13.56 MHz and its nominal power was set to 150 W. The plasma was ignited in the capacitive mode (E-mode).Samples were treated in plasmas created in different gasses: oxygen (O 2 ), ammonia (NH 3 ), or sulphur dioxide (SO 2 ).The pressure was set to 50 Pa.Samples were treated in the afterglow 20 cm away from the coil.Treatment time was 10 s. 2.3.Surface Characterization.Surface functionalization of plasma-treated samples was determined by X-ray photoelectron spectroscopy (XPS).XPS characterization was performed using an XPS (TFA XPS, Physical Electronics, Munich, Germany).The samples were excited with monochromatic Al K 1,2 radiation at 1486.6 eV over an area with a diameter of 400 m.Photoelectrons were detected with a hemispherical analyser positioned at an angle of 45 ∘ with respect to the normal of the sample surface.XPS survey spectra were measured at a pass energy of 187 eV using an energy step of 0.4 eV, whereas high-resolution spectra were measured at a pass energy of 23.5 eV using an energy step of 0.1 eV.An additional electron gun was used for surface neutralization during XPS measurements.All spectra were referenced to the main C1s peak of the carbon atoms, which was assigned a value of 284.8 eV.The measured spectra were analyzed using MultiPak v8.1c software (Ulvac-Phi Inc., Kanagawa, Japan, 2006) from Physical Electronics, which was supplied with the spectrometer.

Cell Adhesion and Morphology Studies.
Human umbilical endothelial cells (HUVEC, ATTC, Manassas, VA, USA) were cultured in MEM medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, USA), 100 U penicillin, 1000 U streptomycin, and 2 mM Lglutamine and plated at density of 3000 cells/cm 2 .For the cell adhesion and morphology investigations, the cells were seeded at a density of 2 × 10 4 cells in 100 L drop of medium on the upper side of the polymers (concentration: 2.55 × 10 4 cells/cm 2 ) and left for 3 h and 24 h to attach at 37 ∘ C in a humidified atmosphere of 5% CO 2 .Cells were seeded onto modified polymer in duplicate for each time and plasma treatment condition.Cells were seeded to the plasma-treated polymer samples within 30 minutes, after plasma treatment.Cell adhesion and morphology were assessed only after 3 and 24 hours of incubation to see differences in adhesion of cells in the first few hours after incubation, because after 24 h cells normally already adapt to different surface conditions and differences are not that pronounced anymore.
Cell adhesion and morphology were investigated by scanning electron microscopy (SEM).Briefly, the polymer samples with the attached cells were fixed in 2% glutaraldehyde (Sigma-Aldrich, USA) in phosphate buffer solution for 5 minutes, followed by dehydration through an increasing gradient of ethanol and then vacuum-dried by the critical point method.Finally the samples were covered by a thin layer of gold and analyzed by SEM.For gold evaporation PECS instrument (Model 682) from Gatan GmbH (Munich, Germany) was used.SEM analyses were performed using a JEOL JSM-840 Scanning Electron Microscope (JEOL, Tokyo, Japan).

MTT Assay.
HUVEC were seeded and cultured in the same manner as for the cell adhesion and morphology investigation by SEM.The MTT-related colorimetric assay (EZ4U; Biomedica, Austria) was used to determine cell growth and viability, according to the manufacturer's instructions.The method is based on the fact that living cells are capable of reducing less colored tetrazolium salts into intensely colored formazan derivatives.This reduction process requires functional mitochondria, which are inactivated within a few minutes after cell death.Briefly, after 3 and 24 hours of HUVEC culture on the differently modified polymer surfaces the medium was removed and the polymer samples were rinsed with phosphate buffer saline to remove for all nonattached cells.Then 200 L of fresh Hanks' Balanced Salt Solution (HBSS) mixed with the tetrazolium agent was added to each well with the polymer sample of the 24-well plate.After 3 and 24 h of incubation, supernatants were transferred into 96-well plates and the absorbance was measured at OD 570/690 nm with Synergy™ HT Microplate Reader (Bio-Tech Instruments, Inc., USA).

Statistical Analysis.
All the above experiments were performed in duplicate and independently repeated at least three times, unless otherwise stated.The results obtained are shown as the mean ± SE for duplicates of cultures.Student's test was used to test the effect different plasma modifications of PCL have on the adhesion and metabolic activity of HUVEC and a value of  < 0.05 was considered significant.

Surface Characterization of Plasma-Treated PCL.
Surface composition of various plasma-treated samples as deduced from XPS survey spectra is shown in Table 1.Each gas used for plasma treatment of the polymer caused different modification of the surface.Oxygen plasma treatment caused oxidation of the PCL surface, where the concentration of oxygen increased from 22 atomic % to 32 atomic %.Contrarily, NH 3 plasma treatment of the PCL samples caused reduction of the amount of oxygen as oxygen concentration decreased to only 15 atomic %.This is due to dissociation of NH 3 in plasma to hydrogen atoms that react with oxygen from the polymer.NH 3 plasma treatment also caused incorporation of nearly 4 atomic % of nitrogen to the polymer surface.
Oxygen concentration on the surface of plasma-treated PCL sample also increased upon SO 2 plasma treatment.Furthermore, 4 atomic % of sulphur was found incorporated to the PCL surface.Oxygen found on the surface of SO 2 plasma-treated PCL sample was bound to sulphur groups rather than carbon atoms as explained later in the text.Additionally, small concentrations of nitrogen were found on the surface of the SO 2 plasma-treated PCL sample.This is not unusual, because many authors have found small amounts of nitrogen on SO 2 plasma-treated surfaces and its presence was explained by traces of nitrogen in the residual atmosphere [30,31].Collaud Coen et al. proposed stabilization of newly formed sulfur group via a hydrogen bond with amine group which can be formed in plasma [30], whereas Holländer and Kröpke proposed reaction (deactivation) of surface radicals with NO which is also formed in the plasma [31].
To get more details regarding surface functional groups induced by various plasma-treatment procedures, we performed deconvolution of high-resolution carbon peaks (Figure 2).Concentration of different chemical groups is shown in Table 2.For untreated PCL polymer (Figure 2 For the case of NH 3 plasma treatment (Figure 2(c)) the intensity of the C-C peak increased, whereas the intensity of other peaks associated with oxygen decreased, which is in correlation with quite low oxygen concentration shown in  2), it is clear that increased oxygen concentration observed in Table 1 is due to the formation of oxygen rich sulphur groups such as C-SO 3 or C-SO 4 .Namely, sulphur S2p peak appeared at approximately 168.6 eV, which is typical for highly oxidized sulphur groups [28].
Because plasma treatment and related surface modification can have significant influence on the surface wettability, we measured water contact angle on different plasma-treated PCL polymers.The contact angle of untreated sample was approximately 110 ∘ .The contact angle slightly increased in NH 3 plasma, whereas in oxygen and SO 2 plasma it decreased significantly and reached approximately 20 ∘ .This means that surface treated in oxygen and SO 2 plasma was very hydrophilic, while the one treated in NH 3 plasma was very hydrophobic.

Cell Adhesion Studies.
Morphology and adhesion of HUVEC on different plasma-treated PCL polymers were investigated with SEM at two time intervals: after 3 and 24 hours (Figure 3).The SEM image after 3 hours of incubation was taken to capture the initial appearance of the cells getting adhered and adapted to the surface.Figure 3 shows that, already after 3 hours, the HUVEC were observed to best adhere to NH 3 -and O 2 -plasma-treated PCL samples, whereas there was nearly no difference between untreated and SO 2 plasma-treated PCL samples.Later, after 24 hours the cells already fully adhered to the surface and the substantial difference in the number of the adherent cells between the untreated sample and plasma-treated samples was observed.Again, the highest number of HUVEC was present on a PCL surface that has been treated with NH 3 and O 2 plasma, which both allowed for significant increase in cell adhesion and likely triggered different cellular signalling pathways, consequently resulting in improved metabolic activity and growth of HUVEC.
Moreover, similar morphology of the PCL samples treated with O 2 and NH 3 plasma, consisting of porous fibrous structure with very thin crisscrossed fibers, was observed by SEM.It is well known that polymer sample morphology plays a crucial role in cell-surface adhesion process [32].Another important effect on cell adhesion has a chemical composition of the surface and surface functional groups, created by plasma treatment [33].Based on the SEM and XPS analyses, the conditions created by O 2 and NH 3 plasma on PCL surface represent optimal condition for cell adhesion and proliferation.Cells exhibit long protrusions, for spreading all over the fibrous surface to cover the maximum surface area for sufficient attachment, which would signal them then to proceed with their proliferation to form the tissue.Thus, great adhesion was achieved after O 2 and NH 3 plasma treatment.The integrity of endothelialization depends on cell-matrix adhesion and the transmembrane proteins called integrins play a crucial role in this process [34].
On the SO 2 plasma-treated PCL samples, the surface morphology changed significantly after plasma treatment.The fibers became denser and thicker, and the surface of the PCL samples appeared less porous.This could be the potential reason for the impaired anchoring of the integrins and subsequently reduced adhesion of cells to the SO 2 treated PCL surface.
With SO 2 plasma treatment sulphur functional groups were introduced to the surface.As reported in the literature by various authors [28,29], sulphur functional groups display antithrombogenic effect by reducing adhesion and activation of platelets.This is extremely important for the production of the prosthetic implants such as synthetic vascular grafts, which are always in direct contact with the blood.However, adhesion of cells to polymer surfaces containing sulphur functional groups does not appear promising, because the SEM images showed poor and impaired adhesion of the cells, although it appears that once the cell is attached to the surface it grows normally.This is supported by the relatively high signal of MTT assay obtained for the SO 2 plasma-treated PCL samples, as explained later in the text.
On the untreated polymer most of the HUVEC appeared shrunken and rounded, with the morphology not typical for HUVEC.Furthermore, a very few attached cells observed on the untreated PCL surface indicate adhesion problems of this surface, which per se possibly represents unfavourable environment for cells.Cells on the untreated PCL surface were not firmly attached to the surface; their morphology changed significantly because of the PLC fibres, which were twisted or captured in between them.
To get additional data on the metabolic activity of the adhered cells and their proliferation on plasma-treated PCL samples, the MTT assay was performed (Figure 4).Three hours after HUVEC seeding no difference in their metabolic International Journal of Polymer Science  activity was observed for SO 2 plasma-treated PCL samples as compared to the untreated one.However, after 3 hours the significant increase of metabolic activity was already observed for O 2 and NH 3 plasma-treated PCL surfaces, compared to control ( < 0.05).After 24 hours, proliferation of HUVEC on these two surfaces increased by more than 60% in comparison to the untreated sample ( < 0.05).Interestingly, polymers treated with SO 2 plasma also showed 40% increase in cell viability as compared to the untreated sample, although SEM images showed poor adhesion of HUVEC to these surfaces.Moreover, after 24 hours the significant differences in cell viability can also be observed in between different plasmatreated samples.One can observe decrease in cell viability on PCL surfaces treated with SO 2 plasma, when compared to NH 3 and O 2 plasma-treated surfaces ( < 0.01).
To summarize, our results clearly show that plasma treatment has an important effect on cell adhesion and viability, as well as cell morphology.NH 3 and O 2 treatment led to strong HUVEC adhesion and viability.HUVEC did also adhere to SO 2 plasma treated-surface; however, their adhesion was poorer and aggravated.Yet, once the cells managed to adhere to the surface they were capable of growing and proliferating further normally.Contrarily, untreated PCL sample represents the least suitable environment for HUVEC adhesion and growth.Enhanced cell proliferation in the case of NH 3 and O 2 plasma treatment was explained by the presence of functional groups that are beneficial for cell proliferation and by different surface morphology.

Conclusions
PLC polymer was treated with NH 3 , O 2 , and SO 2 plasma in order to change surface properties of the sample (i.e., morphology, chemistry, and roughness).We were seeking for the best modification of the PCL surface that would allow for the best cell adhesion and proliferation.Plasma treatment led to changes in surface chemical composition and morphology as well as related hydrophobicity of the polymer, reflecting different adhesion characteristics of cells to the polymer surface.According to literature, the best cell proliferation is normally observed at moderate hydrophobic polymers.Our results show that surface morphology and surface chemistry can be even more important than surface hydrophobicity, because none of our plasma-treated surfaces was moderately hydrophobic.Using various plasmas allowed for incorporation of different functionalities to the surface: various carbon-oxygen functional groups after O 2 plasma treatment, formation of nitrogen groups after NH 3 plasma treatment, and oxidized sulphur groups after SO 2 plasma treatment.MTT assay showed that modification of polymers with O 2 and NH 3 plasmas significantly increased cell adhesion and viability as compared to the untreated polymer, proving that the characteristics of NH 3 and O 2 plasma-treated surfaces are the most favourable for cell adhesion and proliferation.Interestingly, increased viability of HUVEC was also observed on SO 2 plasma-treated surfaces; however, SEM images of SO 2 plasma-treated surfaces showed unfavourable conditions for HUVEC adhesion.Lower cell adhesion can be attributed to changed fibrous morphology of the PCL samples, after treatment in SO 2 plasma.Nevertheless, once the cells managed to adhere to this surface, they were capable of growing and proliferating further normally that indicated the high signal of our MTT assay for SO 2 plasma-treated PCL surfaces.

Figure 2 :
Figure 2: Carbon high-resolution peaks for various plasma-treated PCL samples.
(a)) we can found C-C (284.8 eV) as well as C-O (286.3 eV) and O=C-O groups (288.8 eV).Concentration of these oxygen-containing functional groups has increased after O 2 plasma treatment and a new peak at 287.3 eV due to C=O groups appeared as well, whereas the C-C peak decreased (Figure 2(b)).

3Figure 3 :
Figure 3: SEM images of plasma-treated PCL samples with adhered HUVEC 3 and 24 h upon seeding: (a, e) untreated, (b, f) treated in O 2 plasma, (c, g) treated in NH 3 plasma, and (f, h) treated in SO 2 plasma.

Table 1 :
Surface composition of plasma-treated samples.

Table 2 :
Concentration of different functional groups for various plasma treated samples ( * in the case of NH 3 treatment).
Figure 4: Effect of chemical surface composition on HUVEC proliferation.Proliferation of HUVEC on plasma untreated and treated PCL polymers determined by MTT assay.Student's -test was used to check the statistical significance.# Statistically significant at  < 0.05 for 3 hours compared with control.## Statistically significant at  < 0.01 for 3 hours compared with control.* * Statistically significant at  < 0.01 for 24 hours compared between O 2 and SO 2 plasma treated samples.* * * Statistically significant at  < 0.001 for 24 hours compared with control and/or between NH 3 and SO 2 treated samples.Mean values (±SE) for the respective duplicates are given.