proliferation and antibacterial properties

Applied Surface Science 360 (2016) 641–651

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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc

Atmospheric-pressure DBD plasma-assisted surface modification of polymethyl methacrylate: A study on cell growth/proliferation and antibacterial properties Fatemeh Rezaei a , Babak Shokri a,b,∗ , M. Sharifian c a

Physics Department, Shahid Beheshti University G.C., Evin, Tehran, Iran Laser-Plasma Research Institute, Shahid Beheshti University G.C., Evin, Tehran 19839, Iran c Faculty of Physics, Science Department, Yazd University, P.O. Box 89195-741, Yazd, Iran b

a r t i c l e

i n f o

Article history: Available online 11 November 2015 Keywords: Cold atmospheric plasma PMMA surface modification Surface analysis ATR-FTIR Cell viability Antibacterial property

a b s t r a c t This paper reports polymethyl methacrylate (PMMA) surface modification by atmospheric-pressure oxygen dielectric barrier discharge (DBD) plasma to improve its biocompatibility and antibacterial effects. The role of plasma system parameters, such as electrode gap, treatment time and applied voltage, on the surface characteristics and biological responses was studied. The surface characteristics of PMMA films before and after the plasma treatments were analyzed by water contact angle (WCA) goniometry, atomic force microscopy (AFM) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Also, acid–base approach was used for evaluation of surface free energy (SFE) and its components. Stability of plasma treatment or aging effect was examined by repeating water contact angle measurements in a period of 9 days after treatment. Moreover, the antibacterial properties of samples were investigated by bacterial adhesion assay against Escherichia coli. Additionally, all samples were tested for the biocompatibility by cell viability assay of mouse embryonic fibroblast. WCA measurements indicated that the surface wettability of PMMA films was improved by increasing surface free energy via oxygen DBD plasma treatments. AFM measurement revealed that surface roughness was slightly increased after treatments, and ATR-FTIR analysis showed that more polar groups were introduced on the plasma-treated PMMA film surface. The results also demonstrated an enhancement of antibacterial performance of the modified surfaces. Furthermore, it was observed that plasma-treated samples exhibited significantly better biocompatibility, comparing to the pristine one. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Polymeric materials are of great interest in various biomedical fields [1]. For instance, acrylic polymers are applied as prosthetic materials in ears, dentures, face prosthesis, breath tube and joints [2]. The most important acrylic polymer is polymethyl methacrylate (PMMA), which has been extensively used in various medical and biological fields including drug delivery, orthopedia, orthodontia and ophthalmology [3,4]. PMMA as a biostable and chemically inert material due to its excellent mechanical properties, low cost, and ease of fabrication and processing is widely used as implant [5–7]. However, it is difficult to find a polymer that meets all the requirements, such as antibacterial performance, biocompatibility,

∗ Corresponding author at: Physics Department, Shahid Beheshti University G.C., Evin, Tehran, Iran. E-mail address: [email protected] (B. Shokri). http://dx.doi.org/10.1016/j.apsusc.2015.11.036 0169-4332/© 2015 Elsevier B.V. All rights reserved.

hydrophilicity, roughness and desirable mechanical and volume properties [8]. Microbial infection is one of the most destructive complications related to implanted biomaterials [9]. Bacterial adhesion to the biomaterial surface is the essential step in the pathogenesis of these infections [10]. On the other hand, the optimal interaction of body cells with the biomaterial surface is necessary for cell attachment, spreading, and proliferation [11], and ultimately influences their specific functions [9]. The surface chemistry and topography, directly affecting surface energy and wettability, play essential roles in antibacterial properties and cell adhesion/proliferation [9,12,13]. Therefore, surface modification is required to elicit favorable biological responses to reduce implantation complications while retains the favorable bulk properties. Surface modifications of polymeric biomaterials can be done by various methods, such as grafting of functional groups, wet chemical treatment, ion irradiation, blending, thin film deposition and plasma treatment. Plasma treatment techniques have advantages

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over other approaches because it has the ability to uniformly modify the surface and a few top monolayers of the material surface without any change in the bulk properties [14,15]. The majority of plasma-assisted technologies are based on low-pressure processes [16,17], where high vacuum equipment is needed to achieve adequate plasma conditions, as elaborated in the scientific literature [18]. However, in recent years, due to the lower operational and maintenance costs and simplicity of the equipment, atmospheric-pressure plasmas in dielectric barrier discharge (DBD) configuration have attracted considerable interest for surface modification [15,19]. These systems are also capable of continuous on-line processing for surface modification [15]. Nevertheless, examples of PMMA surface modifications in such configurations, especially for biomedical applications, are not abundant [20]. Previous study by the author [3] has revealed that the surface properties of PMMA and its antibacterial activity can be modified by radio frequency oxygen plasma processing. Recently it was shown that the surface chemistry and structure of a range of polymers can be modified using an atmospheric-pressure DBD plasma [21–24]. Miao and Yun [25] applied air DBD plasma to inactivate Escherichia coli on the surface of Polyethylene terephthalate (PET), Polytetrafluoroethylene (PTFE) and Polyvinyl chloride (PVC). Perni et al. [26] reported the effect of cold atmospheric He-O2 plasma treatment on Polyethylene (PE) in context of total joint and disk replacement. D’Sa et al. [27] studied the effect of air DBD plasma processing on protein adhesion and cell response. Also, enhancement of surface and adhesion properties of polyimide using filamentary and homogeneous air DBDs was achieved by Shao et al. [28]. In this in vitro study, we report on the use of a cold atmosphericpressure oxygen DBD plasma system employed to modify PMMA surface properties. The resultant changes in surface hydrophilicity, energy, morphology and chemical functional groups are investigated. The effects of these changes on both antibacterial activity and cellular response are also studied. E. coli ATCC 25922 and mouse embryonic fibroblast cell (MEF) were used to investigate antibacterial behavior, biocompatibility, and cytotoxicity of samples, respectively. Obtained results demonstrated that atmospheric-pressure oxygen DBD plasma treatment promotes both the biocompatibility and the antibacterial properties of the PMMA surface. 2. Experimental details 2.1. Sample preparation and surface treatment All experiments were performed on PMMA (C5 H8 O2 )n films with dimensions of 1 cm × 1 cm × 0.1 cm. Samples were rinsed successively with deionized water and ethanol using an ultrasonic

cleaner, and finally were dried. The plasma modification was performed via an atmospheric-pressure DBD plasma system composed of two parallel plate aluminum electrodes with 5 cm diameter. The DBD apparatus used in this study is shown schematically in Fig. 1. The lower electrode was covered by a carbonic Teflon (PTFE) as a dielectric barrier (thickness of 1 mm) to limit the current and to prevent the formation of spark and arc discharge. The upper electrode was connected to an AC high-voltage power supply (50 Hz), while the bottom electrode was grounded. Samples were placed on the grounded electrode and the discharge was ignited by applying the AC high-voltage in the range of 15–31 kV (peak-to-peak) at 50 Hz. Oxygen (99.99% purity) was used as the carrier gas. In all experiments, gas flow rate was fixed at 500 sccm (standard cubic centimeter per minute). Oxygen flow rate was controlled with a digital mass flow controller (APEX). In order to ensure the homogeneity and purity of the gas, the plasma chamber was purged for 7 min with O2 gas before treatment. The discharge was then initiated and monitored with a digital oscilloscope (Tektronix DPO3012) to measure the discharge voltage and current. The effect of the electrode gap (2–4 mm), treatment time (5–90 s), and applied voltage (140–300 V) on the surface properties of PMMA samples was studied. 2.2. Surface characterization The chemical functional groups of the surface of PMMA samples were determined by Fourier transformed infrared (FTIR) spectroscopy in attenuated total reflectance (ATR) mode using a Bruker Tensor 27 spectrometer. Accessory for ATR uses single reflection with angle of incidence 45◦ . FTIR spectra were recorded in spectral range of 4000–400 cm−1 with the resolution of 4 cm−1 and 100 scans. To investigate wettability after DBD plasma treatments conducted on the PMMA samples, static water contact angle (WCA) measurements using 3 ␮l volume of distilled water were performed by the sessile drop method [29] at ambient humidity and temperature. All measurements were done 30 min after the treatment in order to allow for all surface relaxation to occur. Also, we applied the van Oss-Chaudhury-Good thermodynamic approach (acid–base theory) to determine the surface free energy (SFE) and its Lifshitz-van der Waals and acid–base components [30]. For this purpose, the contact angle of three liquids, deionized water, diiodomethane (Merck-98%), and formamide (Merck-98%), was measured. The images of each droplet placed on pristine and modified surfaces were processed by ImageJ software. The stability of plasma surface treatment effects, so-called “aging effect”, was also investigated. For aging analysis, WCA measurement was repeated in a period of 9 days after treatment. Samples were stored in ambient air condition and at room temperature.

Fig. 1. Schematic view of the DBD set-up used for PMMA surface modification.

F. Rezaei et al. / Applied Surface Science 360 (2016) 641–651

Tapping mode atomic force microscopy (AFM: DME, Dual Scope C-26 controller) was used to characterize the morphological changes induced by DBD plasma process on a scanned area of 10 ␮m × 10 ␮m. Furthermore, surface roughness, including root mean square roughness (Rrms ) values, was determined. In all experiments, at least three replicates were obtained for each sample and corresponding average values and mean deviations were calculated.

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for 4 h. Afterwards, the remaining solution was removed and 100 ␮l of DMSO was added to wells to dissolve the formazan crystals. Cell viability was evaluated by measuring absorbance (optical density) at 570 nm using a plate reader (ELISA Plate Reader). Obtained optical density (OD) for each well is proportional to the number of living cells [31]. Viability of the cells exposed to samples was calculated using the following equations: Cytotoxicity (%) =



1−

2.3. In vitro antibacterial assay

ODs ODc



× 100

Viability (%) = (1 − Cytotoxicity) × 100 The antibacterial performance against E. coli ATCC 25922 (Gram negative) was investigated by using the modified plate-counting method. For this assay, as described previously [3], the control and modified samples were first washed with 70% ethanol to kill any bacteria on the surface. Then, they were placed in a 3 cm3 solution of bacteria with 0.08 optical absorption coefficient at the wavelength of 600 nm (OD600 nm = 0.08 absorbance = 1.5 × 108 CFU ml−1 ). After that, the samples in the bacterial solution were incubated for 18 h at a temperature of 37 ◦ C. Afterwards, they were thoroughly washed with 2 cm3 of a 0.9% NaCl solution and with 1 cm3 of a 0.05% Tween 20 solution, respectively. Subsequently, 0.01 cm3 of the washing solution was added into different dishes containing the nutrient agar. After 48 h of incubation at temperature of 37 ◦ C, under similar conditions, the colonies of bacteria were counted and the antibacterial activity was quantitatively calculated using the following relationship: B(%) =

C − M  C

× 100

(1)

where B is antibacterial activity (%), C is mean number of bacteria on the control samples and M is mean number of bacteria on the modified samples. Four replicates were considered for each sample. 2.4. Cell culture Mouse embryonic fibroblast (MEF) cells were used for the evaluation of cell adhesion and proliferation. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 supplemented with 10% (v/v) Fetal Bovine Serum (FBS), penicillin (100 U/ml), streptomycin (100 ␮g/ml), and l-glutamine (2 mM). Cells were maintained in a cell culture incubator at 37 ◦ C and in a humidified atmosphere containing 5% CO2 for 24 h. Prior to the experiments, all samples were sterilized by 70 vol. ethanol for 20 min and rinsed three times with sterile phosphate-buffered saline (PBS). MEF cells (1 × 104 cells/well density) were plated in culture medium and left to grow on the sterilized PMMA samples (6.5 mm in diameter and 1 mm in thickness) placed into 96-well plates. Samples in cell suspension were incubated for 48 and 72 h at 37 ◦ C in a humidified incubator with 5% CO2 atmosphere. The investigation of cell adhesion was performed with control (cultural medium with cells) and sample (cultural medium with cells in contact with the polymeric samples) groups. 2.5. In vitro MTT cell viability assay Cell viability of samples was examined using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT method is based on the fact that living cells are capable of reducing less colored tetrazolium salts into intensely colored formazan derivatives. After 48 and 72 h incubation, the culture medium was aspirated off and samples were rinsed twice with 500 ␮l of PBS to remove unattached cells. The remaining cells attached to samples were detached by trypsin-EDTA. Subsequently, 0.1 ml of MTT solution (0.5 mg/ml) was added to each well and cells were then incubated at 37 ◦ C in a humidified 5% CO2 atmosphere

(2) (3)

where ODs and ODc are the average optical density of sample group and control group, respectively. Also, the morphology of cells was examined by using an optical microscope (OLYMPUS CKX41). This assay was carried out in quadruplicate to confirm results. 2.6. Statistical analysis The statistical analysis was performed using OriginPro 8 SR0 software (v8.0724, OriginLab Corporation, USA). One way analysis of variance (ANOVA) was used, with P-values <0.05 considered as significant. 3. Results 3.1. FTIR spectroscopic studies ATR-FTIR spectroscopy was used to study the change in chemistry of the surfaces. The typical ATR-FTIR spectra of pristine and two selected modified samples are shown in Fig. 2(a). Several characteristics peaks of PMMA were found in the spectra. Hummel et al. [32] reported that PMMA compounds can be easily distinguished by bonds in the range of 1260–1150 cm−1 involving C O C stretching mode. The observed frequency at 1482, 1436 and 1387 cm−1 has been assigned to the deformation modes of the methyl (CH3 ) group [4]. The rocking modes, which are depend upon the environment of the CH3 group, are observed at 912 and 841 cm−1 . The bonds at 1190 and 1064 cm−1 are due to the CH3 wagging and twisting, respectively [4]. Further, the bond at 2950 cm−1 can be explained owing to the methylene (CH2 ) asymmetric stretching vibrations. The weak bond appearing at 841 cm−1 has been identified as CH2 rocking vibration [33,34]. The C O and C O bonds often dominate the FTIR spectrum [35]. The C O stretching vibration bond of carbonyl ester group usually lies in the region from 2000 to 1500 cm−1 . As can be seen in Fig. 2(a), PMMA shows a high intensity bond at 1724 cm−1 due to the presence of C O stretching. The C O stretching frequency is one of the intense bonds that appear in the finger print region. This frequency is rather variable and often appears within 1400–1000 cm−1 . The bond at 1144 cm−1 is attributed to the presence of C O stretching mode [5]. Additionally, the C O in plane and out of plane bending is assigned to a medium strong bond at 750 cm−1 . Also, a broad bond for the O H stretching vibration modes of the carboxylic groups (COOH) is also observed in the spectra. This broad bond, as is obvious in Fig. 2(a), appears in the region 3050–2800 cm−1 , centered at about 3000 cm−1 . This is also the region for C H stretching bonds. Therefore, the carboxylic acid shows a somewhat “messy” absorption pattern in this region, with the broad O H bond superimposed on the sharp C H stretching. The O H deformation is in the region 1450–1385 cm−1 and 960–910 cm−1 , although the 1450–1385 cm−1 bond may not be distinguishable from C H bending bonds in the same region. These values agree well with those of Kathiresan et al. [4] and Zwarich et al. [36]. The IR spectra presented in Fig. 2(a) supports the occurrence of the chemical modification of the samples treated by oxygen DBD

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(a)

1.0 0.9

Intensity (a. u.)

0.8 CH2 0.7

O−H and C−H stretches

0.6 0.5 0.4 0.3

C−O stretch

Pristine PMMA C=O stretch

30 s - 260 V

(1724)

30 s - 220 V

C−O−C

0.2 500

1000

1500

2000

2500

3000

3500

4000

-1

Wavelength (cm )

(b)

1.0

(c)

0.9

0.70

0.65 C−O stretching band

C=O carbonyl ester stretching band

0.60

T (%)

T (%)

0.8

0.7

0.55

0.6 0.50

Pristine PMMA 30 s - 260 V 30 s - 220 V

0.5

0.4 1700

1710

1720

1730

1740

1750

0.45

Pristine PMMA 30 s - 260 V 30 s - 220 V

1120 1125 1130 1135 1140 1145 1150 1155 1160 1165 -1

-1

Wavelength (cm )

Wavelength (cm )

Fig. 2. (a) Typical transmittance FTIR spectra of pristine and two selected plasma-modified samples in 4000–400 cm−1 range. Modified-samples correspond to 30 s–260 V and 30 s–220 V plasma treatments. (b) Change in FTIR peak intensity of C O bond (1724 cm−1 ). (c) Change in FTIR peak intensity of C O bond (1144 cm−1 ).

plasma. During plasma treatment, the reactive species in plasma react with the surface of PMMA, resulting in the change of surface chemistry [14]. In Fig. 2(a), intense absorption at 1724 cm−1 has confirmed the presence of C O stretching bond of the carboxylic acid in the polymer structure. ATR-FTIR results generally showed that this peak increased after plasma treatment (Fig. 2(b)). Increasing the intensity of C O bonds could correspond to the formation of carboxylic acid (COOH) [37]. The presence of C O bonds and O H allows us to think that COOH could have been attached on the surface [37]. Also, an increase in the intensity of C O bond is observed after treatment (Fig. 2(c)). As a result, oxygen DBD plasma treatment causes an increase in surface oxygen-containing functional groups. 3.2. Hydrophilicity, hydrophobic recovery and SFE of samples The surface hydrophilicity of PMMA samples is pointed out before and after oxygen DBD plasma treatment in terms of surface WCA. In Table 1, the contact angle values depending on the electrode gap are reported. The WCA of pristine PMMA is 68.7 ± 1.1◦ , indicating that it was reduced after plasma treatment. The lowest

Table 1 Dependence of water contact angle of PMMA films on the electrode gap (treatment time: 30 s, voltage: 220 V). The electrode gap (mm) Water contact angle (◦ )

2 47.2 ± 1

3 42.2 ± 0.7

4 48.4 ± 0.4

contact angle corresponding to maximum hydrophilicity is related to the 3 mm electrode gap (Table 1). Hence, in all subsequent experiments the electrode gap was kept constant at 3 mm. The effect of treatment time and applied voltage on the WCA of samples is presented in Fig. 3(a) and (b), respectively. It is observed that increasing treatment time leads to a reduction of the contact angle. This descending trend continues until 30 s, in which the WCA is 42.2 ± 0.7◦ . After that, the WCA increases with exposure time until it reaches 48.3 ± 0.6◦ , for which the exposure time is 45 s. This increase in contact angles at higher exposure times occurred because excessive treatment of PMMA in O2 DBD plasma increases chain scission, generating a boundary layer that is detrimental to surface hydrophilicity [38]. However, when the treatment continues, the WCA does not change anymore with exposure time. This suggests that there is a saturation effect of plasma treatment [39]. Treatment time could affect the plasma dose in plasma surface processing [5]. According to the FTIR results (Fig. 2), increasing treatment time leads to an increase in the concentration of oxygencontaining functional groups, especially the carboxyl group, on the surface which causes a reduction in the WCA and improvement of surface wettability [14]. Applied voltage is another important parameter for surface treatment, because it can significantly affect the discharge intensity and the energy of the active species [5]. After treatment, the WCA decreases, compared to the untreated PMMA (Fig. 3(b)). It is because the activated species in the plasma environment react with the PMMA surface and produce radicals on the surface layer. The radicals cause the PMMA surface layer to be oxidized [14,40].

F. Rezaei et al. / Applied Surface Science 360 (2016) 641–651

(a)

(a)

75

Surface free energy (mN/m)

ο

Water contact angle ( )

70 65 60 55 50 45 40 -10

0

10

20

30

40

50

60

70

80

90

645

65 60

γAB S

55

γLW S

50

γS

45 40 35 30 25 20 15 10 5 0

100

Pristine

T5

Exposure time (s)

(b)

(b)

70

Surface free energy (mN/m)

ο

Water contact angle ( )

65 60 55 50 45 40 35 0

35

70

105

140

175

210

T15

T30

T45

T60

T90

Experimental condition

245

280

315

65 60

γAB S

55

γLW S

50

γS

45 40 35 30 25 20 15 10 5 0

Pristine

Applied voltage (V)

V140

V180

V220

V260

V300

Experimental condition Fig. 3. Variation of surface WCA of PMMA samples with DBD plasma (a) exposure time in 220 V and (b) applied voltage in 30 s.

As a rule, all the oxidative processes involve an increase of oxygen groups on the treated polymer surfaces, leading to a reduction in the WCA (which was confirmed by means of ATR-FTIR diagnostic) [37,41]. The WCA reaches to its minimum value at 180 V (37.5 ± 0.1◦ ). Further, the effectiveness of oxygen DBD plasma treatment was determined by analyzing surface free energy (SFE). For this purpose, the contact angles of three test liquids (deionized water (w), diiodomethane (d), and formamide (f)) on the sample surfaces were recorded. Information related to these test liquids is given in Table 2. To determine SFE ( s ) and its Lifshitz-van der Waals ( LW ) and acid–base ( AB ) components, the van Oss-ChaudhuryGood thermodynamic approach is used [30]. The latter component  is equal to 2  +  − , in which  + and  − refer to the acidic and

Table 2 Test liquids and their surface tension components [43]. No.

1 2 3

Liquid

Deionized water Diiodomethane Formamide

Surface tension data (mN/m) lLW

l−

l+

l

21.8 50.8 39.5

25.5 0 39.6

25.5 0.01 2.28

72.8 50.8 58.2

Fig. 4. Change in SFE and its components as a function of (a) exposure time in 220 V (T5, T15, T30, T45, T60 and T90 for 5, 15, 30, 45, 60 and 90 s, respectively) and (b) applied voltage in 30 s (V140, V180, V220, V260 and V300 for 140, 180, 220, 260 and 300 V, respectively).

basic constituents, respectively. The following system of equations is used for this analysis [30]:

⎧

 ⎪ − + ⎪ LW  LW + ⎪  s+ w + s− w = 0.5w (1 + cos w ) w ⎪ S ⎪ ⎪ ⎪

⎨   LW  LW +

s+  − +

s−  + = 0.5d (1 + cos d )

S d d d ⎪  ⎪ ⎪ ⎪

⎪ ⎪ ⎪ SLW fLW + s+ f− + s− f+ = 0.5f (1 + cos f ) ⎩

(4)

The effect of treatment time and applied voltage on the SFE of samples is presented in Fig. 4(a) and (b), respectively. Clearly, after plasma treatment the SFE of samples increases. After 30 s oxygen DBD plasma treatment, total SFE and its acid–base component increased, resulting from introducing more polar groups on PMMA surface by plasma [16,42]. When treatment time went on increasing from 30 s to 90 s, these values gradually decreased with the increase of the WCA. There are no significant changes in the Lifshitz-van der Waals component of SFE before and after treatments. It is obvious that the changing trends of total SFE are in accordance with variations of the SAB component. A similar result is also observed for SFE variation with applied voltage. Before

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(a) 60

5s 15 s 30 s 45 s 60 s 90 s

ο

Water contact angle ( )

54

48

Exposure time (s)

5 15 30 45 60 90

42

36

Average roughness (nm) 6.1 7.3 8.1 9.2 10.8 12.5

± ± ± ± ± ±

0.5 0.1 0.1 0.2 0.3 0.1

Applied voltage (V) 0 140 180 220 260 300

Average roughness (nm) 5.2 6.9 7.8 8.1 9.1 10.1

± ± ± ± ± ±

0.4 0.3 0.4 0.1 0.2 0.2

30

3.3. Morphological characteristics

24 0

3

6

9

12

Storage time (day)

(b) 55 140 V 180 V 220 V 260 V 300 V

50 ο

Water contact angle ( )

Table 3 Surface Rrms values of PMMA samples before and after oxygen DBD plasma treatment as a function of exposure time at constant voltage (220 V) and applied voltage at constant time (30 s), respectively.

45

40

35

30

25 0

3

6

9

12

Storage time (day) Fig. 5. Time evolution of the WCA after treatment depending on (a) exposure time in 220 V and (b) applied voltage in 30 s.

Surface roughness is one of the factors influencing surface processes. Therefore, in this study the values of root mean square roughness Rrms are calculated from 10 ␮m × 10 ␮m AFM images. Fig. 6 presents the typical two-dimensional AFM images of PMMA samples under various experimental conditions. These results showed that oxygen DBD plasma treatment could change the surface topography of PMMA. Pristine samples are relatively smooth (Rrms 5.2 ± 0.4 nm), however, some spots emerged on the surface after plasma treatment. These results suggest that PMMA is roughened by treatment, which may be caused by surface etching effects and surface oxidative reactions of plasma treatment [16]. Since the powered electrode is made of aluminum the observed selective etching of the surface may also be regarded to the sputtering effect of the electrode, which would cause deposition of small quantity of metal ions on the surface, leading to selective etching and roughness formation. The obtained Rrms values are summarized in Table 3. As is observed, by increasing treatment time and voltage, surface roughness slightly increases, which, as mentioned above, can be attributed to the surface etching effects being dominant for longer treatment times and higher voltages of the plasma process [16]. 3.4. Antibacterial properties

treatment, total SFE of PMMA was 42.2 mN/m and its acid–base component was 1.2 mN/m. After treatment at 180 V and 30 s experimental condition, SFE increased to 50.6 mN/m and its acid–base component was 7.8 mN/m in this case. Since oxygen plasma treatment enhances incorporation of polar functional groups on the surface, the SAB component is changed considerably, while the SLW component alters negligibly. As expected, increasing total SFE refers to increasing the SAB component. A commonly observed and troublesome phenomenon in plasma treatment is the deterioration of the beneficial surface properties like hydrophilicity with aging [44]. Study of aging effect or “hydrophobic recovery” is important for plasma-modified polymers, especially for biomedical applications. To evaluate if the surface hydrophilicity induced by plasma treatment is durable in time, aging analysis was performed by repeating the WCA measurements in third, sixth and ninth days after treatment (Fig. 5(a) and (b)). It has been suggested that aging effect is mainly due to two processes: (1) reorientation of polar chemical groups toward the bulk of the material to reduce the surface energy, and (2) diffusion of polar chemical groups in the polymer matrix [45,46]. In some cases, the relative reduction in contact angle with time is observed that may be due to oxygen capture from ambient air and creation of more polar surfaces.

E. coli at a cell suspension concentration of 1.5 × 108 CFU ml−1 was used to determine the antibacterial effect of samples. The results are summarized in Fig. 7(a) and (b). The pristine PMMA was taken as a reference in comparing the antibacterial activity values for each sample. It is obvious that the modified samples have an appreciable antibacterial performance compared to the control (pristine) sample. The surfaces T30 and V180 exhibit the highest antibacterial performance against E. coli, implying that the oxygen-containing functional groups have positive effect on reducing bacterial adhesion. 3.5. MEF cell adhesion and growth In order to evaluate the biocompatibility of the plasma-modified samples, MEF cell line was employed to characterize cell adhesion and growth on the surface. About 1 × 104 MEF cells were seeded onto the surface of samples. Following incubation for 48 h, the cells were observed to proliferate considerably. The MEF cell line exhibited different adhesion and growth ability, although it adhered to all sample surfaces. To further study cell growth, MEF cells were added to the samples and subsequently incubated for 72 h. The results of MTT cell viability assay are shown in Fig. 8(a) and (b).

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Fig. 6. Typical two-dimensional AFM micrographs of pristine and treated PMMA samples.

As can be seen, modified surfaces induced more cell adhesion and growth compared to pristine PMMA. However, the quantity of MEF cells on pristine PMMA was poorer than that on the modified samples, but still more than that on the control sample. The cell viability measurements reveal that O2 DBD plasma treatment can significantly promote fibroblast attachment and growth on the PMMA surfaces. 4. Discussion The main objective of this work is to study the biological response of PMMA films after oxygen DBD plasma treatment. It is known that the presence of loosely bond low molecular weight polymer products, produced in the plasma treatment process, may affect the bacterial adhesion and biocompatibility of oxidized polymer materials by causing changes to ionic constitution of a cell culture medium [14,47,48]. Therefore, biological responses of a biomaterial is determined by its surface properties [49]. Hydrophilicity plays an important role in cell and bacteria behavior. The results of WCA measurements disclose that the contact angles are reduced after treatment, from about 68.7 ± 1.1◦ (untreated) to 37.5 ± 0.1◦ (Fig. 3). That is to say, the modified samples yield better wetting properties compared to the pristine sample. This can be attributed to the change of the morphological and chemical properties on the surface after treatment.

In DBD plasma, free electrons are accelerated on the dielectric layer and the kinetic energy of these electrons is high enough to set off basic reactions like excitation, ionization and fragment of the gas species [5,50,51]. During oxygen DBD plasma treatment, active species penetrate the top few nanometers of the film [52]. With the break of C C and C H bonds on the PMMA surface, carbon radicals are produced from the dissociation bonds on the surface, and oxygen-containing functional groups such as COOH and O H are introduced into the PMMA surface [5]. The high concentration of COOH groups at the surface led to reduction of the WCA of PMMA samples (Fig. 9). Also, the C O bonds are strongly hydrophilic, so an increase in its intensity leads to obtain a hydrophilic surface [5]. Therefore, increasing in wettability after plasma treatment is in agreement with ATR-FTIR results, as reported in Fig. 2. Surface roughness is another important factor impacting on cell and bacteria behavior, and thus AFM is utilized to determine the surface morphology. The AFM images acquired from samples (Fig. 6) show that surface morphology changes after treatment, and Rrms values slightly increase (Table 3). Higher roughness which is attributed to some etching during plasma treatment, favoring the anchorage of carboxylic groups [53]. This phenomenon may affect cell and bacteria behavior [38]. However, intensive surface etching is not desirable for biomedical application because the degradation products can lead to the formation of a weak boundary layer on the treated surface which can have a detrimental effect on applications where good adhesion is required [54].

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60

(a) 0.8

*

50

0.7

40

0.6

*

*

48 h 72 h

*

*

* OD at 570 nm

Antibacterial activity (%)

(a)

30 20

0.5

0.3

0

0.2 T5

T15

T30

T45

T60

*

0.4

10

Control

*

ControlPristine T5

T90

T15

T30

T45

T60

T90

Experimental condition

Experimental condition 0.9

*

0.8

50

*

48 h 72 h

40 30 20

*

*

0.7

OD at 570 nm

Antibacterial activity (%)

(b) 60

(b)

*

0.6

* 0.5

*

0.4 0.3

10

0.2

0 Control Pristine V140

Control

V140

V180

V220

V260

V300

Experimental condition Fig. 7. Results of the antibacterial activity of samples against E. coli as a function of (a) treatment time in 220 V (T5, T15, T30, T45, T60 and T90 for 5, 15, 30, 45, 60 and 90 s, respectively) and (b) applied voltage in 30 s (V140, V180, V220, V260 and V300 for 140, 180, 220, 260 and 300 V, respectively). The star (*) indicates statistically significance at P < 0.05 for the modified surfaces vs. the pristine one (control).

A comparison of antibacterial effect (Fig. 7) and wettability results (Fig. 3) confirms that increasing hydrophilicity leads to an enhancement of the antibacterial performance. Surface roughness is the other factor affecting bacterial adhesion. Based on the statistical analysis, Fig. 10 indicates that the relationship between antibacterial activity and the WCA can be expressed by an exponential model (R2 = 0.98), while the relationship between antibacterial activity and surface roughness can be expressed by a linear model (R2 = 0.88). It is observed that the optimum antibacterial performance is obtained at highest hydrophilicity, while an increase of surface roughness deteriorates the antibacterial performance. It is well known that cell walls of most bacterial strains are negatively charged [55,56]. Therefore, more negative surfaces construct an initial defense line against microbial adhesion by an electrostatic repulsion [57]. Oxygen plasma treatment can introduce negatively charged functional groups to the surface [58]. With increasing the concentration of these groups, the surface becomes more hydrophilic and more negatively charged. Thereupon due to the strong repulsive forces, the antibacterial effect increases. Also, as can be seen in Fig. 10, fewer bacteria adhere to the PMMA films

V180

V220

V260

V300

Experimental condition Fig. 8. MTT cell viability assay data for MEF after 48 and 72 h in culture as a function of (a) treatment time in 220 V (T5, T15, T30, T45, T60 and T90 for 5, 15, 30, 45, 60 and 90 s, respectively) and (b) applied voltage in 30 s (V140, V180, V220, V260 and V300 for 140, 180, 220, 260 and 300 V, respectively). The star (*) indicates statistically significance at P < 0.05 for the pristine and modified surfaces vs. the control one.

with lower roughness values because generally rougher surfaces offer higher surface area for attachment. Oxygen DBD Plasma-treated PMMA samples showed an increase in cell viability and proliferation (Fig. 8). Obtained experimental data revealed that some functional groups created by O2 plasma impact the growth of MEF cells positively. This phenomenon illustrates the importance of the surface chemical structure on cell behavior. A comparison of MTT assay data (Fig. 8) with the results of WCA measurements (Fig. 3) implies that enhancing the surface hydrophilicity leads to an increase in cell attachment, which is accompanied by enhancing biocompatibility. The most hydrophilic surfaces have higher biocompatibility. This is due to the production of oxygen-containing functional groups on the surface. Introduction of new functional groups to the surface, such as hydroxyl (O H) and carboxyl (COOH) groups, by oxygen DBD plasma treatment leads to improve biocompatibility [59]. Moreover, it is accepted that hydroxyl groups contribute to cell colonization [49]. The MEF cells adhere and grow after incubating. Typical representative optical micrographs are shown in Fig. 11(a)–(c). It can be seen that for the plasma-modified surface (Fig. 11(c)) the cells form dense structure on the surface. This is not observed

F. Rezaei et al. / Applied Surface Science 360 (2016) 641–651

649

Fig. 9. Images of water droplets onto PMMA surfaces in the ImageJ environment where the variation of the WCA after treatment is observed: (a) pristine PMMA, WCA ∼ 69◦ , (b) DBD plasma-treated sample (15 s–220 V), WCA ∼ 47◦ and (c) DBD plasma-treated sample (30 s–180 V), WCA ∼ 37◦ .

52 13 50

11

46

10

44

9

42

8

40 Water contact angle Roughness

38

Roughness (nm)

ο

Water contact angle ( )

12 48

7 6

36 0

10

20

30

40

50

60

Antibacterial activity (%) Fig. 10. Antibacterial activity dependence on contact angle and surface roughness.

for the unmodified sample (Fig. 11(b)) where we can still find empty places not covered by the cells. These results indicate that the unmodified surface is not optimal for cell adhesion, since the cells even after long incubation time did not completely adapt to the surface. In the case of the plasma-modified surface the cells managed to adapt to it after 72 h of incubation and then good proliferation is observed. These observations confirm that the plasma treatment of PMMA films has improved their biocompatibility for fibroblast cells. A comparison of cell adhesion (Fig. 8) and wettability results (Fig. 3) confirms that increasing hydrophilicity would lead to an enhancement of cellular response. Surface roughness also affect cellular adhesion. Fig. 12 indicates that the relationship between cell adhesion and the WCA can be expressed by an exponential model (R2 = 0.98), while the relationship between cell adhesion and surface roughness can be expressed by a linear model (R2 = 0.82). According to these observations one can conclude that surface roughness affects more the cellular response, indicating that the surface chemistry is mostly responsible for maximizing cell viability.

Fig. 11. Typical optical microscopy images of PMMA surface after 72 h of incubation with MEF cells for (a) control sample, (b) pristine sample and (c) plasma-modified sample (the magnification factor of the images: 100 ␮m).

650

F. Rezaei et al. / Applied Surface Science 360 (2016) 641–651

Acknowledgment

52 13 50

This work was financially supported by Iran National Science Foundation (INSF).

48 11

46

10

44

9

42

8

40 38 36 0.50

7

Water contact angle Roughness 0.55

0.60

0.65

Roughness (nm)

ο

Water contact angle ( )

12

0.70

0.75

0.80

6 0.85

OD at 570 nm Fig. 12. Cell viability dependence on contact angle and surface roughness.

5. Conclusion An atmospheric-pressure oxygen DBD plasma was applied to modify PMMA surface characteristics for biomedical applications. Samples were exposed to reactive oxygen plasma, under various exposure times and applied voltages. The results of the topographical investigations, performed by AFM technique, illustrated that oxygen DBD plasma treatment slightly roughens the surface of PMMA films. The Rrms values of the plasma-treated samples increased from the etching effect during treatment. The decrease in contact angles revealed that oxygen DBD plasma exposure renders the films surface hydrophilic. The WCA analysis showed that short treatment times (30 s) are sufficient to make the surface hydrophilic and introduce desirable functionality to the surface. Aging analysis demonstrated that WCAs increase with the duration of the storage time. According to this data one can say that, the first hours after DBD treatment are the most appropriate for surface involved applications including, thin film deposition, painting, etc. The treated surfaces were found to be chemically modified. Analysis of the ATR-FTIR spectra from the pristine and the plasma-treated samples showed that DBD plasma treatment led to introduction of oxygen-containing functional groups to the surface. Surface modification and formation of new functional groups on the surface contributed to the enhancement of cell viability and antibacterial effects. Mouse embryonic fibroblasts attach and proliferate considerably on the modified substrates (cell growth for optimum condition after 72 h incubation: 171.5%). It is found that more hydrophilic surfaces are able to positively influence cell viability. These hydrophilic surfaces also simultaneously inhibit bacterial adhesion to PMMA surface. The antibacterial activity against E. coli reached to 51.5%. It maybe O2 DBD plasma treatment alone had relatively little effect on the antibacterial properties of the treated samples. According to statistical studies, in order to obtain better cell adhesion and antibacterial effect, proper roughness and morphology were more useful than a higher one. Such conditions were obtained during 30 s plasma treatment at lower applied voltage (optimum condition: 30 s-180 V). So, the DBD plasma treatment process is simple and presents attractive characteristics since it does not require expensive equipment, and moreover, it works at atmospheric pressure and ambient temperature. In addition, no environmentally dangerous byproducts are created as checked through FTIR spectrum recording.

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