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Cytocompatibility of polyhydroxy butyrate modified by plasma discharge.

INTRODUCTION

Synthetic polymers or systems based on polymers find application in biomedicine and tissue engineering. The polymers are advantageous for their favorable properties (easy processing, chemical inertness, etc.). However, particular choice for a certain polymer substrate is often dictated by physico-chemical properties of polymer surface. Not quite suitable properties of pristine polymers (e.g. hydrophobicity) can be improved by chemical or physical modification of their surface [1], The techniques for the polymer surface modification [2] should preserve their favorable bulk properties. By the modification such polymer surface properties as crystalline fraction, surface polarity-wettability, adhesion or adsorption of various agents, printability, chemical reactivity, or light sensitivity can be affected in desirable manner. Preliminary change of polymer surface properties is often a necessary step for subsequent polymer grafting with nanoparticles [3], metal coating [4], or grafting with biologically active species [5]. Some of modification techniques create specific functional groups on the polymer surface [6-9].

The techniques for surface modification [9-12] fall into two main groups. The wet chemical methods are based on the alteration of substrate surface by direct contact with specific reagent. The functional groups or covalent bond with different macromolecular chains of biogenic materials are introduced in this way [7, 9]. The composition of specific reagent significantly influences the physico-chemical properties of polymer surface. By grafting appropriate chemical groups or macromolecules can be attached to polymer surface. For example, the thiol group (SH) can significantly alter both surface biocompatibility [6] and adhesion of gold nanoparticles and subsequent gold layer growth.

Important and often applied physical technique of the surface modification is based on exposure of polymer surface to plasma and corona discharge [13]. The main advantage of plasma treatment is absence of wett chemistry [14], The energy of plasma particles is sufficiently high to disruption of surface bonds, leading to degradation of polymer macromolecular chains. The bond breakage is thermodynamically unstable and the radical sites on the polymer surface easily interact with gas molecules or fragments from outside environment. In this way, new oxygen containing groups may be created on the polymer surface and the surface polarity may increase. Also ablation near surface polymer layers is observed [15]. By the plasma treatment only a very thin surface layer is affected and the polymer bulk remains untouched [14, 16, 17]. The changes in the polymer surface properties depend strongly on plasma discharge power and exposure time [18]. The plasma discharge leads to the creation of chemically active sites (e.g., free radicals) on the polymer surface. The polymer surface exited by plasma discharge relaxes and gradually achieves energetically more advantageous state (thermodynamic equilibrium) [19]. During aging period spontaneous changes in the properties of the plasma-modified polymer are observed, which can also be affected by other external factors (light, temperature, presence of oxygen, etc.).

In this work the surface properties of polyhydroxybutyrate (PHB) pristine or modified by Ar plasma discharge are studied using different techniques. Surface polarity, morphology, and roughness are determined using goniometry and AFM. Thickness of the surface layer ablated by the plasma discharge and/or chemical etching is determined by gravimetry. Chemical composition of the PHB surface is obtained from XPS spectra. The adhesion and proliferation of NIH 3T3 (mouse fibroblasts) on selected samples of the plasma-treated PHB is investigated too.

EXPERIMENTAL

Materials and Modification

PHB (with 8% poly(hydroxyvalerate), density 1.25 g [cm.sup.-3], upper working temperature 95[degrees]C) in the form of 50-pm-thick foils (supplied Goodfellow) was used. The samples were modified in diode plasma discharge on Balzers SCD 050 device for 0-240 s, using DC Ar plasma (gas purity was 99.997%, power 3, 5, and 8 W). Process parameters were: Ar flow 0.3 1 [s.sup.-1], Ar pressure 10 Pa, electrode area 48 [cm.sup.2], the inter-electrode distance of 50 mm, chamber volume 1000 [cm.sup.3].

Measurement Techniques

Contact Angle and Surface Free Energy. Contact angle (CA) was determined by goniometry using static water drop method. The measurements of the advancing water CAs (error [+ or -] 5%) were performed using distilled water on eight different positions using the Surface Energy Evaluation System (SEE System, Advex Instruments, Czech Republic). By automatic pipette the water drop of volume (8.0 [+ or -] 0.2) [micro]l was deposited on the polymer's surface and the consequent photo was evaluated. Estimation of surface energy was also based on the measurement with the SEE System. Two liquids (water and glycerol) were used for the measurements. The CAs were determined using Owens-Wendt method. The measurement was carried out at room temperature.

Surface Morphology. Surface morphology and roughness of the pristine, plasma-modified and/or etched PHB samples were examined by AFM technique using a VEECO CP II device in tapping mode. A Si probe RTESPA-CP, with the spring constant of 20-80 N [m.sup.-1] was used. The mean roughness value ([R.sub.a]) represents the arithmetic average of the deviations from the center plane of the sample.

Surface Chemistry. The presence of the oxygen and carbon in the modified PHB surface layer was proved by X-ray photoelectron spectroscopy (XPS). An Omicron Nanotechnology ESCAProbeP spectrometer was used. The exposed and analyzed area had a dimension of 2 x 3 [mm.sup.2]. The X-ray source was monochromated at 1486.7 eV and the measurement was performed with the step of 0.05 eV. Characteristic Ols and Cls peaks were observed. The spectra evaluation was carried out by CasaXPS software.

Gravimetry. Mean thickness of the removed surface layer after the plasma ablation was measured using a Mettler Toledo UMX2 microbalance. In order to enhance the sensitivity of the measurement the samples were exposed to the plasma from both sides. The thickness of the removed layer was calculated from the average change in weight of six samples before and after the plasma treatment. The depolarization high-frequency gate was used to discharge the sample surface in order to minimize the influence of surface electrostatic charge on the measurement. After weight-loss determination the ablated thickness was calculated from the weight of the ablated layer, area of the sample, and the PHB bulk density. The mass loss caused by plasma treatment combined with etching was also determined. The samples were treated in plasma (8 W, 40-240 s) and than they were put into methanol or water for 21 hours. After etching process the removed samples were dried in BINDER oven at 70[degrees]C for 1 hour and then weighed. The thickness of the removed layer was calculated from the measured change in weight of six samples before and after the plasma treatment and etching.

Cell Adhesion and Proliferation

For cell culture experiments the following adherent cell line was used: mouse embryonic fibroblasts (NIH 3T3) obtained from the German Resource Centre for Biological Materials (DSMZ, Germany). NIH 3T3 cells were cultivated on a regular basis in high glucose Dulbecco's modified Eagle medium with stable 2 mM L-Glutamine (DMEM, PAA, Austria) supplemented with 10% fetal bovine serum (Invitrogen, USA), 1% MEM vitamins solution (Invitrogen, USA), and mixture of antibiotics and antimycotics (100 U [ml.sup.-1] penicillin G, 100 [micro]g [ml.sup.-1] streptomycin sulfate, 0.25 [micro]g [1.sup.-1] amphotericin B; provided by Sigma, USA). The cultures were propagated at 37[degrees]C in 95% humidified air atmosphere with 5% C[0.sub.2]. The cells were maintained in exponential growth and transferred to new tissue culture plates every second or third day.

The bio-response of individual types of PHB matrices was also tested. The polymer samples were first sterilized in glass tubes in 70% ethanol for at least 1 h, air-dried, inserted into 12-well plates for cell cultures (TPP, Switzerland; internal diameter of 2.14 cm), and weighted by poly(methyl methacrylate) cavus cylinders (Zenit, Czech Republic) with the diameter of 2.12 cm. This way prepared foils were seeded with NIH 3T3 cells with the density of 14,000 cells [cm.sup.-2] in 800 [micro]l of complete cell culture medium. The same batch of cells was used for appropriate control, which means cells growing on polystyrene Petri dish (PS) or polystyrene well bottoms of the cell culture plates used on a regular basis. All the samples were plated in triplicates and cells were grown under determined conditions.

Cells intended for analysis by fluorescent microscopy were fixed and stained as previously described. After medium aspiration, the cells were washed twice with phosphate-buffered saline (PBS, pH = 7.4) preheated up to 37[degrees]C. The rinsed cells were fixed on the surface of the substrates by 1 ml of 4% formaldehyde (Thermo Scientific, USA) solution in PBS at 37[degrees]C for 20 min. Phalloidin-tetramethylrhodamine B isothiocyanate (Sigma, USA) solution in PBS (1 [micro]g x [ml.sup.-1]) was used to visualize precisely the filamentous F-actin in cell cytoskeleton corresponding to the cell shape. The staining proceeded in dark at laboratory temperature for 10 min. Cellular nuclei were stained by solution of 4',6-diaminido-2-phenylindole dihydrochloride (DAPI, Sigma, USA) in PBS (0.5 [micro]g x [ml.sup.-1]) in dark at laboratory temperature for 5 min. Between and after the single staining, the cells were rinsed twice by PBS to remove the excess of unbound dyes.

RESULTS AND DISCUSSION

Surface Physico-Chemical Properties

Contact Angle and Aging. The dependence of CA and surface free energy on exposure time by at different discharge powers was determined by standard goniometry (Fig. 1). The measurement was performed immediately after the plasma modification. The average CA of pristine PHB is 66[degrees]. Exposure to lowest plasma discharge power of 3 W leads to the fast decrease of CA already after 10 s of treatment. For 40 s exposure CA achieves a minimum value of about 30[degrees]. For exposure times above 40 s CA increases slowly with increasing exposure times. This turnabout in the CA dependence may be because of the surface ablation and change in the surface roughness [20]. The similar trend is observed at higher discharge power 5 W where the lowest CA (36.6[degrees]) is observed for exposure time of 10 s. For longer exposure times CA increases again. At 8 W discharge power monotonous but slow CA decrease with increasing exposure time is seen. This behavior is little bit surprising as on different synthetic polymers studied before CA decreases monotonously up to very long exposure times, the decrease being initially fast and slower for longer exposure times [21, 22]. Plasma treatment could lead to cleavage of macromolecular chains, creation of free radicals, and conjugated double bonds [23]. Reaction with oxygen from ambient atmosphere in reaction chamber or after exposition of the samples to atmosphere results in production of oxygen-containing polar functional groups (e.g. hydroxyl, carbonyl, carboxyl, peroxide, and ester groups) [23],

Surface free energy, estimated from CA, as a function of the plasma discharge power and exposure time is shown in Fig. IB. The surface energy of pristine PHB is relatively low (ca 39 [m.sup.-2]). Plasma treatment results in a sharp increase of the surface energy for both discharge powers of 3 and 5 W. For exposure times above 50 s the surface free energy decreases and achieves saturated value of 50 mJ [m.sup.-2] for the 240 s exposure time. At 8 W discharge power the surface free energy increases over whole exposure time range from 10 to 240 s and achieves maximum (60 mJ [m.sup.-2]) for the exposure time of 240 s.

The effects of sample aging and surface ablation upon plasma treatment and chemical etching were studied on the samples plasma exposed at highest discharge power of 8 W. The dependence of the CA on the aging time for the samples modified at the discharge power of 8 W for 10 and 240 s is shown in Fig. 2A. Immediately after the plasma treatment very fast increase in CA is observed for both exposure times. On PHB plasma treated for 10 s CA increases monotonously and after 72 hours it achieves saturation value of about 67[degrees], which is close to that of pristine PHB. The aging curve of the PHB plasma treated for 240 s is quite different. After initial rapid increase CA increases further but much more slowly and it achieves saturated value of about 93[degrees] after 170 hours of aging. This value is much higher than that of pristine PHB. The aging time evolution of CA is connected with spontaneous rearrangement of degraded macromolecules and creation of new functional groups on the plasma-treated surfaces [5]. Corresponding dependence of the surface free energy is shown in Fig. 2B. On PHB modified at the discharge power of 8 W for 240 s initial fast decrease of the surface energy is seen followed by an increase up to a saturated value. For shorter exposure time of 10 s no such pronounced dependence on the aging time is found. Instead the free surface energy fluctuate in this case especially for the shorter exposure times.

Mass Loss of Treated Surface. The mass loss because of the plasma treatment and chemical etching was examined on the PHB modified at 8 W discharge power for the times from 40 to 240 s. The dependence of the thickness of removed polymer layer on the plasma exposure time is shown in Fig. 3. As could be expected the removed thickness is an increasing function of the exposure time. By plasma discharge about 86-nm-thick surface layer is removed after exposure for 240 s. Subsequent etching in water and methanol results in removal of substantial amount of material from the sample surface. Etching in water was chosen because the biocompatibility tests, described below, are performed in water environment. Etching in methanol may be of interest for possible grafting of plasma-treated PHB with bioactive agents. The thickness loss up to 260 nm was observed for plasma-treated PHB (8 W and 240 s) and then etched with water. The etching of plasma-treated PHB in methanol is much more intensive and the thickness loss achieves 5000 nm in this case. The etching is supposed to remove primarily low weight oxidized structures created by the plasma treatment on the polymer surface [20].

Surface Morphology and Chemistry. The surface morphology and roughness was determined on the pristine PHB ([R.sub.a] = 3.2 nm) and the plasma-treated and chemically etched PHB and typical AFM images are shown in Fig. 4. The plasma treatment induced an increase of the surface roughness ([R.sub.a] = 8.1 nm). The etching in water leads to minor decrease of surface roughness ([R.sub.a] = 7.0 nm), but the surface morphology remains almost unaffected. Methanol etching causes a slight increase in the surface roughness ([R.sub.a] = 9.5 nm), which may be caused by more intensive etching process (see Fig. 3). Despite of rather strong etching of the plasma-treated PHB only a mild change both of surface morphology and roughness on such treated samples was observed.

The XPS method was used for the determination of the surface chemistry of pristine and plasma-treated PHB (discharge powers 3 and 8 W, exposure time 240 s). The samples were measured immediately after the plasma modification and after the aging time, at which the CA achieves constant, saturated value (see Fig. 2). The atomic concentrations of oxygen are shown in Fig. 5. The surface concentration of oxygen on pristine PHB is 28%. The plasma treatment with 3 W leads to a mild decrease in oxygen concentration. In this case the degradation of oxygen containing groups overcomes their creation. The aging does not result in a significant change in the oxygen concentration. On the PHB samples modified at 8 W discharge power the oxygen concentration increases immediately after the plasma treatment and the oxygen concentration increases further during the aging period. For the PHB sample modified at 8 W discharge power the oxygen concentration of 33% is achieved. The increase of the oxygen concentration on the PHB surface during aging is probably caused by spontaneous reorientation of polar groups towards the sample surface and oxygen adsorption from ambient atmosphere [24]. The observed changes in the oxygen concentration on the PHB surface may also be affected by the auto-oxidation processes [25] involving fluctuation of proxy, hydroxyl, and carbonyl groups during aging period. The amount of oxygen on PHB aged surface modified with 8 W and 240 s was determined to be 33%.

Surface Cytocompatibility

The influence of PHB plasma treatment on mouse embryonic fibroblasts (NIH 3T3) cytocompatibility is documented in Figs. 6 and 7. It is evident, that the proliferation and growth of NIH 3T3 cells is satisfactory even on pristine PHB, the results being even better than on standard tissue PS (Fig. 6). The plasma activation of PHB leads to further improvement of PHB surface biocompatibility manifested in cell proliferation and growth (third day from cell seeding). The result is significantly better in comparison with pristine PS as a comparative material. The images illustrating the cells proliferation and growth on pristine and plasma-modified samples are shown in Fig. 7. The results on pristine and plasma-modified PHB (8 W discharge power, 240 s exposure time) are compared with those obtained on tissue PS. It is obvious, that plasma activation enhances the cell proliferation only mildly, but the cell homogeneity is significantly improved (Fig. 7H). The plasma-modified PHB exhibits even higher cell number and better homogeneity in comparison with tissue PS.

CONCLUSION

The treatment of PHB in Ar plasma leads to a decrease of the measured CA (increase of wettability). The differences in CA and surface energy during the aging of the plasma-modified PHB, however, the CA increases and after some time it achieves a saturated, constant value. Both effects depend strongly on the plasma discharge power. The exposure to the plasma discharge leads to ablation of PHB surface layer, the thickness of which is an increasing function of the plasma exposure time. Subsequent etching of the plasma-modified PHB in water and methanol results in additional removal of PHB surface layer. The methanol appears to be much more effective etching medium. In the case of methanol etching surface layer ca 5000 nm thick was removed. Surface ablation by more plasma treatment is much less effective, it results in removal of PHB surface layer, the thickness of which is by about two orders of magnitude smaller. The plasma modification changes PHB surface chemistry. Namely, the oxygen concentration on the PHB very surface is changed, the change being dependent on the plasma discharge power and the sample aging time. Most pronounced increase in the oxygen concentration is observed on aged PHB, plasma modified at 8 W discharge power. The plasma modification influences positively PHB cytocoinpatibility. The proliferation, adhesion, growth, and spreading of mouse embryonic fibroblasts (NIH 3T3) on the plasma-modified PHB are even better than on tissue PS.

ACKNOWLEDGMENTS

This work was supported by the GACR under projects 108/10/1106 and P108/12/1168.

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P. Slepicka, (1) S. Styblova, (1) Slepikova Kasalkova, (1) Rimpelova, (2) V. Svorcik.

(1) Department of Solid Engineering, Institute of Chemical Technology, 166 28 Prague, Czech Republic

(1) Department of Biochemistry and Microbiolagy, Institute of Chemical Technology, 166 28 Prague, Czech Republic

Correspondence to: P. Slepicka; e-mail: petr.slepicka@vscht.cz

DOI 10.1002/pen.23666

Published online in Wiley Online Library (wileyonlinelibrary.com).
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Author:Slepicka, P.; Styblova, S.; Kasalkova, N. Slepickova; Rimpelova, S.; Svorcik, V.
Publication:Polymer Engineering and Science
Article Type:Report
Geographic Code:4EXCZ
Date:Jun 1, 2014
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