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Materials Research

Print version ISSN 1516-1439

Mat. Res. vol.16 no.4 São Carlos July/Aug. 2013  Epub Apr 23, 2013

http://dx.doi.org/10.1590/S1516-14392013005000056 

Benefits of oxygen and nitrogen plasma treatment in Vero cell affinity to poly(lactide-co-glycolide acid)

 

 

Andrea Rodrigues EspositoI, II, *; Camila Mika KamikawaI; Carolina LucchesiI; Betina Mara Pereira FerreiraI; Eliana Aparecida de Rezende DuekI, II

ILaboratory of Biomaterials, Center for Medical and Health Sciences, Pontifical Catholic University of Sao Paulo, Sorocaba, SP, Brazil
IIDepartment of Materials Engineering, Faculty of Mechanical Engineering, State University of Campinas, Campinas, SP, Brazil

 

 


ABSTRACT

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. 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.

Keywords: biomaterials, plasma treatment, PLGA, cell culture


 

 

1. 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 regenerate1.

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 tissues2. 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 tissues1.

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 function3. 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 wettability4, surface charge5, roughness and topography6. Hence, surface modification techniques have been used to improve surface properties of polymers7.

Biodegradable polyester, including poly(lactide-co-glycolide) (PLGA) with various lactide/glycolide ratios, is an important material for tissue engineering8, despite its poor hydrophilicity and lack of natural cell recognition sites on its surface9.

Plasma treatment is an effective and widely used method for modifying the surface of materials10,11. Moreover, reactive sites such as amine12, sulfonic acid and carboxyl groups13 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 polymers14.

Plasma processes have been used to increase the hydrophilicity of polylactide (PLA) and improve its cell adhesion12. Santos Junior et al.15 and Ferreira16 studied the adhesive force and affinity of Vero cells to PLLA and PHBV films, demonstrating that these polymers have poor cell-matrix interaction16. 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 films17.

In a comparative study, Croll et al.17 and Hasirci19 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.

 

2. Material and Methods

2.1. 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: N2 or O2 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 ISO10993-118.

 

Table 1. Parameters for PLGA treatment with O2 and N2 plasma.

 

2.2. 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.

2.3. 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.

2.4. 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% CO2 at 37 °C. Vero cells, a recommended line of cells, were used for cytotoxicity and cell-substratum interactions with biomaterials studies1,15,20-23.

2.5. 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 assay23. After incubation, 2 × 105 cells.mL - 1 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-diphenyl-tetrazolium 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 (DMSO, 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. ANOVA was used to perform comparisons of continuous variables of all groups and Tukey's test was used to compare the groups (p < 0.05).

2.6. Cell morphology analysis - SEM

In order to carry out cell morphology analysis approximately 2 × 105 Vero cells.mL - 1 of medium Meio 199 (EARLE) supplemented with 10% FCS were seeded in each well containing the membranes and cultured at 37 °C, 5% CO2. 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 (Blazersï CDT 050). The coated specimens were observed with a JEOL 5800 scanning electron microscope.

2.7. Cytochemical analysis

For the cytochemical study 2 × 105 cells.mL - 1 were cultured on PLGA membranes in medium Meio 199 (EARLE) supplemented with 10% FCS maintained in a gas-jacket incubator with 5% CO2 at 37 °C. After 2, 48 and 168 hours in culture, the samples were fixed with Karnovsky fixative (4% paraformaldehyde, 2.5% glutaraldehyde in 0.2 M phosphate buffer, pH 7.2), dehydrated in graded ethanol series at room temperature, cleared in xylene and stained with Toluidine Blue (TB) at pH 4.0, a basophilic dye, for the detection of DNA, RNA and glycosaminoglycans and with Xylidine Ponceau (XP) at pH 2.5, an anionic dye, for identification of cationic proteins15. Staining time was 15 minutes for TB and 20 minutes for XP, both at room temperature. The samples obtained were photographed under a Nikon Eclipse E800 photomicroscope.

 

3. Results and Discussion

3.1. 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 (γsp) of the surface energy.

 

*Ys surface energy, Ysp = polar component, Ysd = dispersive component.

Table 2. Effects of O2 and N2 plasma treatment on PLGA surface energies.

 

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 surfaces24-26. 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.

3.2. 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 process27, 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 roughness28.

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 scaffolds1,16,29-31. 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). On membrane surfaces where cells will likely complete their movement and adhere tightly, it was observed the development of focal contacts within a short cultivation time32. 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 cells33 (Figure 3).

 

Figure 2. The SEM images show Vero cells on different PLGA membranes cultured for 2 hours. a) PLGA (0), b) PLGA O2 (1), c) PLGA O2 (2), d) PLGA N2 (3), e) PLGA N2 (4). White scale bar represents 10 µm.

 

 

Figure 3. The SEM images show Vero cells on different PLGA membranes cultured for 48 hours. a) PLGA (0), b) PLGA O2 (1), c) PLGA O2 (2), d) PLGA N2 (3), e) PLGA N2 (4). White scale bar represents 10 µm.

 

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).

 

Figure 4. The SEM images show Vero cells on different PLGA membranes cultured for 7 days. a) PLGA (0), b) PLGA O2 (1), c) PLGA O2 (2), d) PLGA N2 (3), e) PLGA N2 (4). White scale bar represents 10 µm.

 

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 charges1,15,35,36. Oxygen 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 proliferation15 as to improve cell adhesion and growth8,9,12,24.

3.3. 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.

 

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).

 

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 N2 (3) and PLGA N2 (4) membranes presented similar results regardless to the positive control (p > 0.05). While, PLGA O2 (1) and PLGA O2 (2) membranes have presented intermediate analogous values independent of treatment conditions (p > 0.05). The results are shown in Figure 6.

 

Figure 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)

 

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 proliferation1,2. 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 proliferation25.

Cell adhesion on biodegradable polyester including PLLA, PLDLA and PLGA with various lactide/glycolide ratios is slow, despite their biocompatibility26, possibly due to their poor hydrophilicity29. 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 Claes37 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 functions2, it was considered that the immediate measurement of the mitochondrial activity in terms of cell sensitivity would be enough to evaluate cell viability.

3.4. 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.

 

Figure 7. Vero cells cultured on different PLGA membranes and stained with Xylidine ponceau (XP): a) PLGA O2 (2) after 2 hours, 200×; b) PLGA N2 (3) after 2 hours, 100×; c) PLGA O2 (1) after 168 hours, 400×; d) PLGA N2 (4) after 168 hours, 100×.

 

 

Figure 8. Vero cells cultured on different PLGA membranes and stained with Toluidine blue (TB): a) PLGA O2 (1) after 48 hours, 200×; b) PLGA N2 (4) after 48 hours, 100×; c) PLGA O2 (2) after 168 hours, 100×; d) PLGA N2 (3) after 168 hours, 100×.

 

According to the results obtained by Santos1,31 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 copolymers30,38.

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.

 

4. Conclusions

Oxygen 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.

 

Acknowledgements

The authors would like to express their appreciation to PIBIC-CNPq for financial support, the Brazilian agency FAPESP (project no. 03/02188-5), the Brazilian Synchrotron Laboratory for giving us permission to use the plasma, SEM, AFM, and XPS equipment, and São Paulo State University, Sorocaba, for the goniometer.

 

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Received: May 31, 2012
Revised: November 6, 2012

 

 

* e-mail: aesposito@fem.unicamp.br

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