Benefits of Oxygen and Nitrogen Plasma Treatment in Vero Cell Affinity to Poly ( Lactide-Co-Glycolide Acid )

Cell adhesion on materials surface is critical because this phenomenon occurs before other events, as cell spreading, cell migration and cell differentiation. It is commonly accepted that the adhesion of cells on solid substrate is influenced by several substratum surface properties, such as wettability, surface charge, roughness and topography. Plasma technique is a convenient method for modifying surface properties of materials without affecting physical properties. In this study, poly(lactide-co-glycolide), PLGA, membranes were modified by oxygen and nitrogen plasma to improve polymer hydrophilicity and verify their effect on Vero cells culture. The PLGA membranes, which were characterized by SEM and contact angle, showed increased surface rugosity and narrower contact angles. Cell adhesion, cytotoxicity assay, SEM and cytochemistry analysis showed that plasma treatment was beneficial to cell growth by improving cell-polymer interaction.


Introduction
A research area that has received increasing attention is tissue engineering that restores tissue functionality from native or synthetic sources by applying engineering principles.Biomaterials play an important role providing scaffolds to guide tissue regeneration, means to deliver drugs and growth factors to stimulate tissue response, or by creating a new functional structure when damaged tissue cannot regenerate 1 .
Whenever biomaterials are employed it is essential to understand their pattern of cell growth and differentiation and select the appropriate polymer, given that the latter may influence the development of bioresorbable polymers designed to stimulate the regeneration of damaged tissues 2 .The importance of cell culture methodology related to biomaterials research is that it enables fast evaluation of the performance of polymers.In addition, evaluation of cell-biomaterial interactions concerning the prediction of possible in vivo reactions to these polymers is invaluable when they are utilized as substitutes for body parts or as a stimulant for regeneration or repair of damaged tissues 1 .
In tissue engineering, cell adhesion onto a surface is critical because it occurs before other events, as cell spreading, cell migration and differentiation of cell function 3 .Moreover, cell adhesion is closely related to surface properties of biomaterials.It is generally accepted that adhesion of cells onto solid substrate is influenced by several substratum surface properties, such as wettability 4 , surface charge 5 , roughness and topography 6 .Hence, surface modification techniques have been used to improve surface properties of polymers 7 .
Biodegradable polyester, including poly(lactide-coglycolide) (PLGA) with various lactide/glycolide ratios, is an important material for tissue engineering 8 , despite its poor hydrophilicity and lack of natural cell recognition sites on its surface 9 .
Plasma treatment is an effective and widely used method for modifying the surface of materials 10,11 .Moreover, reactive sites such as amine 12 , sulfonic acid and carboxyl groups 13 on polymers (i.e.functional surfaces) can be created by using non-polymerizing gases.It has been shown that plasma processes can only proceed with localized surface treatment that does not change the bulk properties of the polymers 14 .
Plasma processes have been used to increase the hydrophilicity of polylactide (PLA) and improve its cell adhesion 12 .Santos Junior et al. 15 and Ferreira 16 studied the adhesive force and affinity of Vero cells to PLLA and PHBV films, demonstrating that these polymers have poor cell-matrix interaction 16 .The characterization study indicated that the surface composition and the functional groups on the surface of PLLA and PHBV after plasma treatment had a great effect on cell affinity of these films 17 .
In a comparative study, Croll et al. 17 and Hasirci 19 studied the plasma etching effect on PLGA.The results showed an increase of hydrophilicity of PLGA membranes.However, there are few reports on PLGA surface morphology after oxygen and nitrogen plasma treatment and the effect of surface morphology on cell adhesion.
Thus, the aim of this study was to investigate the effect of oxygen and nitrogen plasma treatment on the PLGA surface properties, including hydrophilicity and morphology, and to evaluate Vero cells affinity to treated PLGA.

Preparation of PLGA membranes
Poly(lactide-co-glycolide), PLGA, was dissolved in methylene chloride (5% W/V) at room temperature.The solution was casting onto glass plates and dried for 24h.Plasma treatment was performed using a radio frequency Anatech LTD Ashing plasma equipment.Table 1 summarizes the plasma treatment conditions: N 2 or D 2 gas, power range of 50-100 W, pressure of 20 to 80 Pa, and time at 600s.The values of power, pressure and time were chosen based on preliminary studies.Previously, before cell culture, the membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates with 100 µL of culture medium and maintained at 37 °C for 24 hours, according to ISD10993-1 18 .

Contact angle analysis
Contact angles were measured in air, using a NRL CA goniometer Ramé-Hart Imaging System, model 100-00, and image acquisition was performed with Ramé-Hart Imaging 2001 software.Demineralized and deionized water and diiodomethane were used as probing liquid in order to obtain contact angles, free energies, and polar and dispersive components of the surfaces.

Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) was used to investigate the morphology of membrane surfaces.The membranes were stuck on a metallic support and coated with gold by using a Sputter Coater Bal-Tec SCD 050.The samples were observed with a JSM 5900-LV ARP scanning electron microscope.

Cell culture
Vero cells, a fibroblastic cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were obtained from Adolfo Lutz Institute, Sao Paulo, Brazil.These cells were cultured in medium Meio 199 (EARLE) (Cultilab  , Campinas, SP, Brazil) supplemented with 10% fetal calf serum (FCS, Cultilab  ) maintained in a gas-jacket incubator with 5% CD 2 at 37 °C.Vero cells, a recommended line of cells, were used for cytotoxicity and cell-substratum interactions with biomaterials studies 1,15,20-23 .

Cell adhesion and viability assays
Identification of the effects of plasma treatments on early cell adhesion and cell viability was carried out using the MTT assay 23 .After incubation, 2 × 10 5 cells.mL - in 100 µL of medium Meio 199 (EARLE) supplemented with 10% FCS were seeded to the wells containing different PLGA membranes treated.The cells were cultured for 2 hours and 24 hours at 37 °C to enable cell adhesion and direct cell viability assays, respectively.Subsequently, the cells were washed twice with 0.1 M phosphate-buffered saline (PBS) pH 7.4 at 37 °C and incubated with 100 µL of medium, MTT assay mixture [10 µL per well, containing 5 mg.mL-1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide -MTT, Sigma  ] was added to each well, and cells were incubated for 4 h at 37 °C.After 4 hours, the culture medium in each well was substituted with 100 µL of Dimethyl Sulphoxide (DMSD, Sigma  ) and 25 µL of buffered Glycine/Sorensen solution to dissolve formazan crystals.The absorbance was quantified spectrophotometrically at 570 nm, using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA).The culture plate itself (polystyrene) and phenol 1% were used, respectively, as the cell viability assay positive and negative controls.Polytetrafluoroethylene dishes (PTFE) and the culture plate itself were used as negative and positive controls for the cell adhesion test.
The absorbance of all experimental conditions was read without cells to control MTT reaction.ANDVA was used to perform comparisons of continuous variables of all groups and Tukey`s test was used to compare the groups (p < 0.05).

Cell morphology analysis -SEM
In order to carry out cell morphology analysis approximately 2 × 10 5 Vero cells.mL - of medium Meio 199 (EARLE) supplemented with 10% FCS were seeded in each well containing the membranes and cultured at 37 °C, 5% CD 2 .As control, cells were cultured on glass cover slips in the same culture conditions.After 2, 48 and 168 hours, the samples were fixed in a fixative solution prepared by dissolving 2.5% paraformaldehyde, 2.5% glutaraldehyde, 0.03% picric acid, 1% tannic acid in 0.1 M cacodylate buffer and the same volume of medium Meio 199 (EARLE) for 1h at room temperature, washed in 0.1 M cacodylate buffer, post-fixed in 1% osmium tetroxide (Sigma  ) in water for 1h at RT in the dark and washed in water.The samples were dehydrated with ethanol series, critical point dried (Balzers  CDT 030) and coated with gold in a sputter coater (Blazers CDT 050).The coated specimens were observed with a JEDL 5800 scanning electron microscope.

Cytochemical analysis
For the cytochemical study 2 × 10 5 cells.mL - were cultured on PLGA membranes in medium Meio 199 (EARLE) supplemented with 10% FCS maintained in a gas-jacket incubator with 5% CD 2 at 37 °C.After 2, 48 and

Contact angle analysis
Treatment of PLGA samples with nitrogen and oxygen plasma drastically narrowed the water contact angle as the result of increased surface hydrophilicity (Table 2).Hence, the notable increase of hydrophilicity on the sample surface is due to the enhancement of the polar component (γ s p ) of the surface energy.
Hydrophilicity is one of the most important factors that influence biomaterial cytochemical compatibility.Cell adhesion and growth on the surface are considered to be strongly dependent on the hydrophilicity/hydrophobicity balance.Some authors have demonstrated that cells are preferentially anchored to hydrophilic surfaces [24][25][26] .The study has also shown that the cells adhere, spread, and grow easier on substrates with moderate hydrophilicity than on hydrophobic or highly hydrophilic substrates.In accordance with Yamaguchi et al. 9 , showed that the majority of the cells presented good cellular adhesion on the surface with contact angles below 60°.
It was also observed that increased hydrophilicity is directly related to augmentation of plasma pressure, power and time (Tables 1 and 2).These results are corroborated by Chu et al. 14 who observed that hydrophilicity is related with the type of gas and treatment parameters.

Scanning Electron Microscopy (SEM)
Changes in the surface morphology are observed in Figure 1 which shows SEM micrographs of PLGA with and without plasma treatment.The untreated PLGA sample (Figure 1a) presents a flat slightly porous surface from solvent casting rate during the manufactory process 27 , while PLGA samples treated had the surface morphology altered by oxygen showed rough morphology (Figure 1b-c) and nitrogen plasma presenting deep holes (Figure 1d-e).There were significant morphological surface changes as the plasma parameters increased.
According to Yang et al. 12 , roughness is one of the reasons for hydrophilicity improvement in the plasma treated samples, given that the surface wettability is consequence  of the surface roughness.Materials of lower surface energy (i.e.polymers) showed an increase in cellular adhesion associated with the increased surface roughness, whereas materials of higher surface energy (i.e.metals) demonstrated little change in cellular adhesion with increased surface roughness 28 .Briefly, physical and chemical properties of the polymer surfaces can be completely modified by plasma treatments.Not only the chemical changes of the surface, but also the topographic changes can contribute independently for modifications in PLGA hydrophilicity.
Cell morphology on treated and untreated membranes was quite different.The initial cell adhesion to the untreated PLGA membrane was poor.The cells were small, rounded and separated from each other (Figure 2a), which is in accordance with other studies using Vero cells seeded on untreated scaffolds 1,16,[29][30][31] .In contrast, it was noticed that Vero cells cultivated for 2 hours on plasma-treated PLGA presented higher density of adhered cells in close contact with each other, spread on the membrane surface.The cells showed flat and elongated morphology and some vesicles on their surfaces (Figure 2b-d).Dn membrane surfaces where cells will likely complete their movement and adhere tightly, it was observed the development of focal contacts within a short cultivation time 32 .After 48 hours in culture, the cells seeded on PLGA membranes were anchored to the substrate and displayed a spread, flattened/elongated morphology with a large number of cells with phillopodes and lamellipods, similar to fibroblastic cells 33 (Figure 3).
According to Berry et al. 34 , fibroblastic cells are sensitive to topography, which affects motility and proliferation.It was also observed processes through which cells were bonded to each other by prolongations and thin filaments budding from the basal portion of the cell (Figure 3c).
Cell-cell contact is necessary for signaling and expression of particular tissue functions.These prolongations often formed a thin fibrillar reticulated material on the cell surface (Figure 3b).
The Vero cells had reached confluence in all samples after 7 days in culture appeared as layers covering the PLGA surface.Clear differences between the pressure and power applied were not observed (Figure 4).
The high number of Vero cells attached on the membranes should be result of cell proliferation in this period, showing that plasma treatment not only supports adhesion but also proper cell division and growth.
A key factor that influences the adherence of Vero cells is the substratum surface charges 1,15,35,36 .Dxygen and nitrogen plasma treatment improved the time span (2 hours)  for interaction between Vero cells and the PLGA membrane by promoting anchorage to the substrate, spreading and proliferation.
This result is in accordance with those in which the surface chemical composition and its topography affect the interaction force that acts between the biomaterial and the biological medium, for instance, water and ion adsorption, protein adsorption, adhesion, cellular expansion and proliferation 15 as to improve cell adhesion and growth 8,9,12,24 .

Cell adhesion and viability assays
Initial cell adhesion assay results showed that Vero cells have presented expected lowest adhesion on the negative control, independent of treatment conditions (p < 0.05).The capacity of cell adhesion to treated PLGA membranes was shown to be similar to untreated PLGA (p < 0.05).All samples presented a low capacity to stimulate cell adhesion when compared with the positive control (p < 0.05).The results are shown in Figure 5.The polystyrene plate itself is commonly accepted as positive control once consists of the same material as the cells are usually culture in vitro, once it allows cell adhesion and proliferation as a gold standard.In the same vein, polytetrafluoroethylene dishes (PTFE) and phenol (1%) represent negative controls once are non-sticker material and toxic, respectively 1,15,22,27,31 .
The cell viability assay results showed that all PLGA membranes are not cytotoxic to Vero cells when compared to the negative control (phenol) (p < 0.05), demonstrating the cytocompatibility improvement of the treatment.PLGA N 2 (3) and PLGA N 2 (4) membranes presented similar results regardless to the positive control (p > 0.05).While, PLGA D 2 (1) and PLGA D 2 (2) membranes have presented  intermediate analogous values independent of treatment conditions (p > 0.05).The results are shown in Figure 6.
Moreover, the positive and negative controls showed significant differences between means (p < 0.01), providing consistent data for standard controls.
MTT assay revealed that all materials in the study did not affect mitochondrial activity of Vero cells.Nitrogen and oxygen plasma proved to be more receptive to cell interaction after 24 hours in culture.
The main cell-biomaterials interaction parameters are toxicity, cell adhesion and proliferation 1,2 .Biocompatibility interaction is very closely related to cell behavior on the receiver recognition of the cell adhesion surface and the connections fields of the extracellular matrix proteins (fibronectin, collagen or vitronectin) that form a compound which can induce cell attachment, spreading and proliferation 25 .
Cell adhesion on biodegradable polyester including PLLA, PLDLA and PLGA with various lactide/glycolide ratios is slow, despite their biocompatibility 26 , possibly due to their poor hydrophilicity 29 .The difference observed in cell viability to PLGA membranes could be related to their chemical composition and topography variation.
As shown in the study conducted by Ignatius and Claes 37 on PLGA biocompatibility, the scaffolds did not present cytotoxic effects, given that the mitochondrial activities of cells were not affected.Although, toxic substances do not act at one specific level but affect several cellular functions 2 , it was considered that the immediate measurement of the mitochondrial activity in terms of cell sensitivity would be enough to evaluate cell viability.

Cytochemical analysis
In the culture period of 2, 48 and 168 hours, we detected cells stained by TB or XP, which are basophilic and acidophilic dyes, respectively (Figures 7 and 8).The cells were able to reach confluence on all PLGA substrates.Cytochemical alterations induced by the different treated and non-treated PLGA membranes on which the cells were cultured were not found.Basophilic cells stained for detection of nucleic acids and glycosaminoglycans with TB pH 4.0 were found in all samples.The basophilic cytoplasm was confirmed by using XP -a stain for protein groups.
According to the results obtained by Santos 1,31 the basophilic cytoplasm suggests that the cells are enabled to activate protein synthesis.These results agree with the description that different cells are able to produce extracellular matrix on PLGA copolymers 30,38 .
The surface modifications were successfully induced to improve cell interaction.The results of Vero cell in vitro culture showed that plasma treatment of PLGA surface provided favorable conditions for cell adherence and spreading due to interaction improvement between the membrane polar groups and the cell surfaces.Consequently, the cells could grow and proliferate naturally in a short lapse of time.Cytochemistry did not show alterations in cell behavior induced by different PLGA treatments.Therefore, it can be concluded that the substrate surface charge greatly influences the growth of Vero cells in adhesive experiments.

Conclusions
Dxygen and nitrogen plasma treatment promoted narrower contact angles and consequently increased hydrophilicity and rugosity of PLGA samples.
Plasma-treated surfaces provide better cellular adhesion, enabling cell growth, spreading and proliferation independently of the gas-type treatment, demonstrating the biocompatibility of the samples.
This work may effectively contribute to the development of biomaterials with different properties and increased feasibility of cell/polymer interaction.

Figure 5 . 6 .
Figure 5. Adhesion of Vero cells cultured on different PLGA membranes after 2 hours.Mean ± sd (n = 6).All treatment results are higher than the negative control (p < 0.05).6.Vero cell viability cultured onto different PLGA membranes after 24 hours.Mean ± sd (n = 6).All treatment results are higher than the negative control (p < 0.05).

Table 1 .
Parameters for PLGA treatment with D 2 and N 2 plasma.

Table 2 .
Effects of D 2 and N 2 plasma treatment on PLGA surface energies.