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Improved cellular adhesion to acetone plasma modified polystyrene surfaces
Journal of Colloid and Interface Science 281 (2005) 122–129 www.elsevier.com/locate/jcis
Improved cellular adhesion to acetone plasma modified polystyrene surfaces S.A. Mitchell, M.R. Davidson, R.H. Bradley ∗ Advanced Materials and Biomaterials Research Center, The Robert Gordon University, School of Engineering, Clarke Building, Aberdeen AB10 1FR, UK Received 24 March 2004; accepted 10 August 2004 Available online 21 September 2004
Abstract The plasma polymerization of acetone has been used to modify polystyrene substrates for the controlled growth of human fibroblast cells. The surface modified polystyrene was studied by X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy. This showed the surface oxygen levels and wettability to increase rapidly with exposure to the acetone plasma. High-resolution XPS allowed the determination of the relative amounts of surface hydroxyl, carbonyl and carboxyl groups. This showed that there was little incorporation of carboxyl groups in the deposited films. AFM measurements revealed the films to be conformal with a surface roughness equivalent to that of the underlying polystyrene substrate with film growth rates of approximately 0.5 nm min−1 . High edge-definition patterns were produced with a simple masking procedure and allowed the confinement of cells to selected areas of the substrate. These chemically patterned surfaces allowed the study of cells confined to particular regions of the substrate as a function of incubation time. 2004 Elsevier Inc. All rights reserved. Keywords: Plasma modification; Polymer surfaces; XPS; AFM; Contact angle; Cell adhesion
1. Introduction The interactions between the biological environment and biomaterials take place at the material–fluid interface. The biocompatibility of a material is governed by the interactions between the implant and biological system on the microand nanometer scale [1–3], and the physicochemical surface properties of the material, e.g., surface chemistry, wettability, surface energy and surface charge [4–8]. The low surface energy of many polymers such as polystyrene and polyethylene, which are essentially nonpolar in their native forms, limits their ability to support cell attachment and growth. This may be due to the adsorption of nonattachment proteins such as albumin. Many surface modification techniques exist for producing surfaces that support cell attachment and a number of these concentrate on fixing extracellular matrix proteins such as fibronectin * Corresponding author. Fax: +44(0)1224-262837.
E-mail address: [email protected] (R.H. Bradley). 0021-9797/$ – see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2004.08.049
and laminin or short amino acid sequences of these proteins to the surface. Plasma polymerization is one technique frequently used to produce chemically functionalized biocompatible surfaces for a range of applications [9,10]. A thin modified surface layer is desirable in most applications because a layer that is excessively thick can alter the mechanical properties of the substrate material and risk delamination. A uniform layer around 1 nm in thickness should be sufficient to determine the wettability of the material and have a profound influence on its biocompatibility. Ideally, the surface modification process should yield films with minimal thickness while accomplishing the required uniformity, durability, and functionality. The attachment and proliferation of mammalian cells is strongly influenced by the physicochemical properties of the surface. In the absence of surface bound ligands or hydrogels the surface free energy is strongly correlated to the rate of cellular attachment. It is generally found that highenergy surfaces promote rapid cellular adhesion and spreading, whereas low energy surfaces do not favor such behavior.
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Since the primary adsorptives in culture media are extracellular proteins [11,12] the reasons for this discrimination are not obvious. It is possible the adsorption of nonattachment proteins such as albumin to hydrophobic substrates is driven by the reduction in liquid–surface interface energy by the displacement of water molecules from the surface [13]. Although the mechanism of cellular adhesion is poorly understood, the marked discrimination between surfaces of differing wettability may be used to regulate the attachment of cells at an interface [14,15]. In recent years, techniques have been developed to produce surfaces with well-defined chemical heterogeneity, which are suitable for this purpose. Surfaces containing regions of high surface energy within a low surface energy matrix are known to adsorb cells in a spatially selective manner. Such spatial control has been successfully demonstrated using patterned self-assembled monolayers (SAMs), typically using a photolithographic or a ‘rubber stamp’ technique [16] and the spatial resolution of this method is sufficient to produce patterns of subcellular dimensions. It has been shown that high resolution patterns of this type can be employed to modify the size and shape of attached cells which influences cell behavior [17–23]. These studies demonstrate the possibility of directing cell growth and behavior through the use of advanced surface engineering techniques. There are many possible applications of devices employing this technology in the study of cell behavior, organization and cellular interactions, as well as in the construction of novel biomedical devices. The major drawbacks of SAMs are their labor-intensive preparation and susceptibility to oxidation [24]. This paper outlines a simple, one-step procedure to produce a well-defined, chemically heterogeneous surface with a subcellular resolution using plasma polymerization. Plasma polymerization for patterning is advantageous, as it is applicable to a wide range of substrates with little or no pretreatment. Additionally a wide range of chemical functionalities may be deposited, albeit with much less specificity than is achievable using SAMs. However, careful control of the plasma conditions enables the production of surfaces with well defined chemistry and wettability. This work demonstrates that plasma polymers may be patterned with subcellular dimensions and highlights some of the factors that may be of importance in achieving high spatial resolution using this approach.
2. Experimental 2.1. Plasma polymerization The internal surfaces of 55 mm diameter polystyrene (PS) dishes (Nunc, UK) were exposed to an acetone plasma in home-built plasma chamber described in detail elsewhere [25]. The RF power at 13.56 MHz was coupled to the chamber via a manual impedance matching network (Coax-
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ial Power Systems Ltd., UK) using an externally wound copper coil. A Pirani gauge and digital controller (Terranova, USA) monitored pressures before and after each deposition. A base pressure of typically 1 × 10−3 mbar was achieved using a rotary vacuum pump (Welch Thomas Vacuum Model 8917C, USA) fitted with a LN2 cooled trap (Kurt J. Lesker, UK). The leak rate was measured before each plasma deposition process and at all times was 0.02 cm3 (STP) min−1 . Analytical grade acetone (Sigma Chemical, Germany) was degassed through several freeze– pump–thaw cycles and introduced into the plasma reactor through a fine control leak valve (LV10K, BOC Edwards, UK). An RF power of 10 W and a constant flow rate of ∼15, ∼44 or ∼56 cm3 (STP) min−1 was used in all experiments with a range of deposition times from 10 to 600 s. Samples for use in cell culture experiments were washed with HPLC grade water (Millipore, resistivity 18 M cm) with gentle agitation for 60 min and dried under aseptic conditions for 120 min. The production of chemically heterogeneous surfaces was achieved using a simple masking technique [26] whereby a copper transmission electron microscope (TEM) grid was placed on the surface of the PS dish and exposed to the plasma. After treatment was complete the TEM grid was gently removed from the surface to reveal the chemically patterned surface. 2.2. Cell culture Human fibroblast cells (1BR.3N) were grown in 75 cm2 / canted neck tissue culture polystyrene, TCPS flasks (Nunc) and incubated at 37 ◦ C under a 5% CO2 atmosphere. Culture media was minimum essential medium (MEM) (Labtech International, Ringmer, UK) with HEPES modification containing 25 mM HEPES, 10% (v/v) Foetal Calf Serum (Labtech International), 1 unit/ml penicillin (Labtech International), 100 µg/ml streptomycin solution (Labtech International), 100 µg/ml nonessential amino acids (Labtech International) and 2 mM L-glutamine (Lancaster Synthesis, UK). Prior to harvesting, cells were rinsed in 2 × 10 ml phosphate buffered saline (PBS) and harvested with 2 ml of Trypsin–EDTA solution (Labtech International) for a maximum of 5 min at 37 ◦ C. The trypsin was deactivated by the addition of 20 ml media culture. After centrifugation for 3 min at 340g, the pelleted cells were gently resuspended in fresh media. For the cell attachment profile experiments, a suspension of cells, ∼10,000 cells/ml, was added to each acetone plasma treated PS dish and untreated control dish. The dishes were left undisturbed at 37 ◦ C in a 5% CO2 atmosphere between observations. To determine their attachment profile the cells were observed at 10× magnification at 24, 48 and 72 h and counted using an overlaid grid with dimensions calibrated using a graticule observed at the same magnification. Chemically patterned surfaces were also observed using the above apparatus after exposure to an atmosphere of ∼95% humidity.
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2.3. X-ray photoelectron spectroscopy (XPS) The surface chemical composition of acetone plasma treated dishes was studied using a Kratos Axis HSi 5 channel imaging X-ray photoelectron spectrometer using monochromated AlKα radiation (1486.6 eV) operated at 150 W in a residual vacuum of <4 × 10−9 mbar with the analyzer in fixed transmission mode. Charge neutralization was used for all samples with the standard operating conditions for insulator surfaces: −2.3 V bias voltage, 1.0 V filament voltage and 1.9 A filament current. All spectra were acquired in hybrid mode, i.e., using both the electrostatic and the magnetic lenses. Elemental surface compositions were calculated from survey spectra measured at a pass energy of 80 eV with the error of the quantification estimated at ±10%, deduced from the analysis of a PTFE standard.
Fig. 1. Surface oxygen obtained when varying the W/FM parameter of an acetone plasma treatment. Error bars represent 1 SEM. The line shown is a guide to the eye.
2.4. Atomic force microscopy (AFM) The topography of acetone plasma treated surfaces was studied using a Digital Instruments (DI) Nanoscope IIIa SPM under ambient conditions. The surfaces were imaged in contact mode using a silicon nitride tip. Image analysis was carried out using DI version 4.23r6 software. Surface roughness (RMS) was calculated from 3 different 20 ×20 µm areas on each treated surface. 2.5. Contact angle (CA) The wettability of washed acetone plasma treated surfaces was assessed by photographing a static 20 µl droplet of doubly distilled water on the surface with a digital camera. The contact angle of the droplet on the surface was measured directly from the image.
3. Results and discussion 3.1. XPS analysis of acetone plasma modified PS The composite parameter (W/FM) is used as a measure of the total energy per unit mass of gas where W, F and M are the RF power, monomer flow rate and molecular weight of the monomer respectively. The parameter W/FM can be used to characterize the plasma conditions during the modification process. Increasing W/FM results in a higher degree of fragmentation and molecular rearrangement whereas lower values of W/FM leads to a greater retention of the chemical functionality of the monomer. The surface oxygen concentration of polystyrene exposed to an acetone plasma for 60 s as a function of the W/FM parameter is shown in Fig. 1. It can be seen that the surface oxygen concentration reaches a saturation value of ∼12 at.% for a W/FM value of ∼0.8. Further increases in W/FM does not lead to increased oxygen levels. Acetone (CH3 COCH3 ) has an oxygen content of 25 at.% so it is clear that with all plasma powers used
some oxygen is lost, probably in the form of volatile species such as CO and CO2 [27]. Several studies using acetone as a monomer have shown it to produce films with very low deposition rates [28]. It should be stressed that the results shown in Fig. 1 are likely to be from films with thickness less than the analysis depth of the XPS technique. However, oxygen can also be introduced to the polystyrene surface via the quenching of free radicals produced by vacuum ultraviolet (VUV) irradiation. The variation in surface oxygen concentration shown in Fig. 1 may reflect differences in VUV intensity produced by plasmas of different flow rates. A previous study by Ertel et al. using plasma polymerized acetone found that the growth of bovine aortic endothelial cells correlated well with the carbonyl content of the films but did not correlate with the concentration of hydroxyl or carboxyl groups [29]. For this reason we used low values of W/FM to minimize monomer fragmentation and to produce films containing a high concentration of carbonyl groups. The surface oxygen concentration of the plasma deposited films with flow rates of ∼15, ∼44 and ∼56 cm3 (STP) min−1 and RF powers of 10 W are shown in Figs. 2a, 2b and 2c respectively. The acetone plasma treatments resulted in an increase in surface oxygen concentration from 0% to (a) ∼17%, (b) ∼8% and (c) ∼12%. For each flow rate there is initially a rapid increase in surface oxygen concentration followed by a more gradual increase. The results shown in Figs. 1 and 2 were obtained from unwashed surfaces. Washing with water resulted in minimal change in the surface oxygen concentration (<2 at.%) which is within the error of the XPS technique. Further information on surface chemistry changes resulting from the deposition process has been obtained by analysis of the carbon 1s peak envelope. An example of the functional group distribution of a PS surface exposed to the acetone plasma for 600 s at a flow rate of ∼15 cm3 (STP) min−1 is shown in Fig. 3. The C 1s peak can be resolved into 6 components: C–C/C–H at 285.0 eV; βC–C/C–H which is βshifted by +0.4–0.6 eV due to the juxtaposition of carbons
S.A. Mitchell et al. / Journal of Colloid and Interface Science 281 (2005) 122–129
Fig. 2. Surface oxygen concentration of the acetone plasma treated PS surfaces. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. Error bars represent 1 SEM. The lines shown are guides to the eye.
Fig. 3. Peak fitted C 1s XP spectrum for a polystyrene surface exposed to the acetone plasma for 600 s with a flow rate of ∼15 Sccm.
bonded to oxygen; C–OR/βC–OR at a shift of +1.6; C=O with a shift of +3.0; O–C=O groups with a shift of +4 eV and a peak due to π–π ∗ shake-up satellites with a shift of +5–8 eV [30–32]. The relative areas of these peaks as a function of treatment time are shown in Fig. 4. This shows that the oxidation process initially proceeds via the formation of C–OR and carbonyl groups up to approximately 120 s exposure. At treatment times over 120 s the concentration of C–OR and C=O groups continue to increase but at a rate less than that of the first 120 s. For a flow rate of ∼15 cm3 (STP) min−1 the concentration of O–C=O groups starts to increase after approximately 120 s, whereas for flow rates of ∼44 and ∼56 cm3 (STP) min−1 the increase begins between 180 and 300 s. The π–π ∗ functionality appears to decrease rapidly
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Fig. 4. Oxygen functional group distribution of acetone plasma treated PS as a function of exposure time. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. (C–O, 2; C=O, "; O–C=O, Q; π –π ∗ , F.) The lines shown are guides to the eye.
within the first 120 s of treatment followed by a more gradual reduction. A reduction of the intensity of this peak, which originates from the aromatic structure of polystyrene, could be due to the deposition of a plasma polymer film or a disruption of the phenyl groups. Studies of UV-ozone oxidation of polystyrene is also known to reduce the intensity of this peak which has been attributed to ring-opening reactions [33–35]. Exposure of polystyrene to short wavelength ultraviolet of the type generated in a glow discharge is known to lead to polymer chain scission with accompanied abstraction of hydrogen and the formation of a number of radical species [36]. In an oxygen-containing discharge, such as an acetone plasma, it is thermodynamically favorable for these radicals to react with gaseous species present in the reaction chamber [37]. However, there may also be a degree of oxygen incorporation due to quenching of residual free radicals on exposure to air. It is likely that a combination of these processes is responsible for the observed oxygen incorporation. 3.2. Contact angle analysis of acetone plasma modified PS The water contact angle of acetone plasma treated PS surfaces with monomer flow rates of (a) ∼15, (b) ∼44 and (c) ∼56 cm3 (STP) min−1 are shown in Fig. 5. The acetone plasma treatments resulted in a reduction in water contact angle from ∼92 to (a) ∼72, (b) ∼70 and (c) ∼77◦ . The reduction in contact angle is to be expected with the deposition of a film containing hydrophilic oxygen species.
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Fig. 5. Water contact angle of acetone plasma treated PS surfaces. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. Showing the increased wettability with acetone plasma treatment time. Error bars represent 1 SEM. The line shown is a guide to the eye.
3.3. Wettability of acetone plasma micropatterned PS Chemically heterogeneous polystyrene surfaces were prepared by exposing surfaces to the acetone plasma with selected areas masked. A 250 mesh TEM grid with a 30 µm bar width masked a PS dish that was exposed to the plasma with a flow rate of ∼15 cm3 (STP) min−1 and 10 W power for 300 s. The dish was exposed to an atmosphere of high humidity (∼95%) to visualize the chemical pattern. Fig. 6 shows the contrast between the untreated regions (area I) and those exposed to the plasma (area II). There is a larger incidence of water droplets on the unmasked, treated, areas with little adsorbed water on the more hydrophobic, untreated areas. 3.4. AFM analysis of plasma treated PS AFM measurements found no systematic variation in surface roughness for any of the flow rates used and the films were found to follow the topography of the PS substrate which has a RMS roughness of between 3 and 5 nm [38]. Deposition depths were determined by lightly scratching the acetone plasma film deposited on a silicon wafer with a scalpel blade and imaging the resulting trenches with the AFM. A similar scratching force to that used on the coated surfaces on an uncoated silicon wafer did not result in the formation of a trench. Film deposition rates obtained for flow rates of ∼15, ∼44 and ∼56 cm3 (STP) min−1 were 0.43, 0.18 and 0.47 nm min−1 respectively. The reduced deposition rate with a flow rate of 44 cm3 (STP) min−1 suggests that a higher degree of ablation or etching is occurring at this flow
Fig. 6. Optical image of micropatterned PS surface that has been exposed to an acetone plasma for 300 s then exposed to an atmosphere of ∼95% humidity for 5 s. Patterning achieved using a 250 mesh transmission electron microscope grid (100 µm pitch, 25 µm bar and 75 µm hole). I, unmasked (treated) area; II, masked (untreated) area. Scale bar = 100 µm.
rate. Etching of polymer surfaces will occur in an oxygencontaining plasma and the etch rate will increase with increasing flow rate. As flow rate increases further the etch rate will reach a maximum and begin to decrease as there is no longer sufficient RF energy to dissociate the monomer to form reactive oxygen species [39]. 3.5. Cell culture on acetone plasma modified PS surfaces The rate of attachment and spreading of human fibroblast cells (1BR.3N) on the acetone plasma treated surfaces was significantly greater than on native PS. Images obtained from regions of a chemically patterned surface show that under otherwise identical culture conditions, cells on the treated areas exhibit a flat morphology indicating relatively strong attachment and spreading, whereas the majority of those on the untreated regions were rounded, a sign indicative of poor attachment. Rinsing the surface with phosphate buffered saline (PBS) removed the majority of cells from the untreated regions which further demonstrated their poor attachment. The proliferation of 1BR.3N cells was monitored on untreated and treated PS surfaces. The fold increase was used as a measure of cell proliferation and is calculated by dividing the number of spread cells after 48 h by the number of spread cells counted after 24 h. Data points marked with an asterisk, in Fig. 7, represent a surface oxygen concentration that shows a significant difference (P < 0.05, by t-test) in fold increase observed between untreated PS (0% surface oxygen) and that of the data point. For comparison, a data point for tissue culture polystyrene is also shown in Fig. 7. Fig. 7 shows the fold increases of 1BR.3N cells grown on acetone plasma treated PS surfaces treated with monomer flow rates of ∼15, ∼44 and ∼56 cm3 (STP) min−1 . For each
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increases in surface oxygen leads to lower proliferation rates although for all treated surfaces the rate of cell growth is significantly greater than that found on untreated polystyrene. Interestingly, for each flow rate, the surface which exhibited the greatest fold increase had no XPS-detectable carboxyl (O–C=O) content, a functional group previously shown by a number of studies to support growth and proliferation of human fibroblasts [27]. This supports the hypothesis that surface wettability rather than the concentration of any particular functional group is the most important determining influence on a materials ability to support cell attachment and proliferation. It has been shown in a number of studies that plasma modified polymer surfaces can enhance the attachment of anchorage-dependent cells such as endothelial and fibroblasts via the adsorption of cell adhesion proteins such as fibronectin and vitronectin [40,41]. The adsorption of these proteins is controlled by a number of factors including: wettability, topography and functional group distribution. The functional group distribution will influence the conformation and composition of the adsorbed protein layer. Protein adsorption onto a substrate is therefore important in controlling cellular interactions with synthetic surfaces in vivo and in vitro in the presence of serum. Since protein adsorption from serum-containing media occurs rapidly the direct recognition of surface functional groups by the cells is virtually impossible and thus functional groups are believed to affect cell adhesion via interaction of the adsorbed protein layer. Therefore, differences observed in cell attachment and growth is most likely due to differences in the adsorption of proteins by the polymer surface. Cell culture surfaces (prepared by gas plasma (glow discharge) methods) are typically chemically complex and contain a variety of chemical functional groups and it is often difficult to determine the effect of any one chemical functionality on attachment and growth. Furthermore, it should be stressed that the findings presented here are for one cell type and it may be the case that different cell types will respond differently to the same modified surface. 3.6. Cellular patterning using acetone plasma modification of PS Fig. 7. Fold increase of 1BR.3N cells on acetone plasma treated surfaces relative to surface oxygen concentration as determined by XPS. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. Error bars represent 1 SEM. The line shown is a guide to the eye. A data point for tissue culture polystyrene (2) has been included for comparison.
flow rate there appears to be a sharp increase in proliferation rates even at very low surface oxygen concentrations. For flow rates of 15 and 56 cm3 (STP) min−1 the maximum proliferation is found with a surface oxygen concentration of ∼5 at.% with fold increases of ∼2 and 16, respectively. For films prepared with a flow rate of 44 cm3 (STP) min−1 the maximum fold increase of ∼7 occurs at an oxygen concentration of ∼1.5 at.%. For all plasma treatments, further
To obtain patterns of cellular resolution the masking technique described above was employed to restrict surface modification to well defined areas of the PS dishes. Fig. 8 shows images of an acetone plasma patterned PS dish that has been seeded with 1BR.3N cells and incubated for (a) 72, (b) 96, (c) 120, (d) 144 and (d) 168 h. The acetone plasma treatment used was at a flow rate of 15 cm3 (STP) min−1 for 60 s using a Sjostrand TEM grid to mask selected areas. At low incubation times the pattern is not clearly defined but as the cells grow it is clear that cells have attached predominately on the unmasked (treated) areas and spread preferentially in the direction of the treated surface resulting in aligned, elongated cells. At extended incubation periods (168 h) the encroachment of cells onto the untreated areas
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(a)
(b)
(c)
(d)
(e)
Fig. 8. 1BR.3N cells grown on acetone plasma patterned PS dish after (a) 72, (b) 96, (c) 120, (d) 144 and (e) 168 h incubation. 60 s exposure, TEM grid used was a Sjostrand mesh. Scale bars = 100 µm.
occurs after the treated zones become confluent. Figs. 8d and 8e show the cells several abreast in the bars of treated surface and have remained partially aligned, presumably due to the spreading constraints on those cells close to the chemical boundary. At present the exact mechanism by which cells spread from cell-friendly oxidized areas onto neighboring hydrophobic areas is not known.
faces composed of both hydrophilic and hydrophobic zones under otherwise identical conditions. At short incubation periods the cells preferentially attached to the hydrophilic, plasma treated areas until confluence was reached. Extended exposure caused spreading of the cells to the untreated, hydrophobic areas. Since cell line and primary cells do not normally attach to untreated polystyrene this finding seems paradoxical and may reflect changes in the cellular environment which warrant more detailed study.
4. Conclusion This study has shown that it is possible to produce surfaces with controllable oxygen chemistry using an acetone plasma. The chemistry and wettability of the surfaces were studied using XPS and contact angle measurements which demonstrated that even short plasma treatments resulted in significant changes to the polystyrene surfaces. Careful control of plasma conditions: exposure time, RF power and flow rate, allowed the production of thin films (thickness <1 nm) with no carboxyl groups. Oxygen levels of up to 17 at.% were found for samples exposed with monomer flow rates of 15 cm3 (STP) min−1 . The exact mechanism of oxygen incorporation is not clear but there are several mechanisms that may be relevant for this system including film deposition, ablation, UV irradiation from the glow discharge, postplasma oxidation after exposure to air, or a combination of these. The deposition rates found with this process were very low at approximately 0.5 nm min−1 allowing the production of chemically patterned surfaces with very abrupt chemical gradients. This allowed the study of cell attachment to sur-
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Improved cellular adhesion to acetone plasma modified polystyrene surfaces S.A. Mitchell, M.R. Davidson, R.H. Bradley ∗ Advanced Materials and Biomaterials Research Center, The Robert Gordon University, School of Engineering, Clarke Building, Aberdeen AB10 1FR, UK Received 24 March 2004; accepted 10 August 2004 Available online 21 September 2004
Abstract The plasma polymerization of acetone has been used to modify polystyrene substrates for the controlled growth of human fibroblast cells. The surface modified polystyrene was studied by X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy. This showed the surface oxygen levels and wettability to increase rapidly with exposure to the acetone plasma. High-resolution XPS allowed the determination of the relative amounts of surface hydroxyl, carbonyl and carboxyl groups. This showed that there was little incorporation of carboxyl groups in the deposited films. AFM measurements revealed the films to be conformal with a surface roughness equivalent to that of the underlying polystyrene substrate with film growth rates of approximately 0.5 nm min−1 . High edge-definition patterns were produced with a simple masking procedure and allowed the confinement of cells to selected areas of the substrate. These chemically patterned surfaces allowed the study of cells confined to particular regions of the substrate as a function of incubation time. 2004 Elsevier Inc. All rights reserved. Keywords: Plasma modification; Polymer surfaces; XPS; AFM; Contact angle; Cell adhesion
1. Introduction The interactions between the biological environment and biomaterials take place at the material–fluid interface. The biocompatibility of a material is governed by the interactions between the implant and biological system on the microand nanometer scale [1–3], and the physicochemical surface properties of the material, e.g., surface chemistry, wettability, surface energy and surface charge [4–8]. The low surface energy of many polymers such as polystyrene and polyethylene, which are essentially nonpolar in their native forms, limits their ability to support cell attachment and growth. This may be due to the adsorption of nonattachment proteins such as albumin. Many surface modification techniques exist for producing surfaces that support cell attachment and a number of these concentrate on fixing extracellular matrix proteins such as fibronectin * Corresponding author. Fax: +44(0)1224-262837.
E-mail address: [email protected] (R.H. Bradley). 0021-9797/$ – see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2004.08.049
and laminin or short amino acid sequences of these proteins to the surface. Plasma polymerization is one technique frequently used to produce chemically functionalized biocompatible surfaces for a range of applications [9,10]. A thin modified surface layer is desirable in most applications because a layer that is excessively thick can alter the mechanical properties of the substrate material and risk delamination. A uniform layer around 1 nm in thickness should be sufficient to determine the wettability of the material and have a profound influence on its biocompatibility. Ideally, the surface modification process should yield films with minimal thickness while accomplishing the required uniformity, durability, and functionality. The attachment and proliferation of mammalian cells is strongly influenced by the physicochemical properties of the surface. In the absence of surface bound ligands or hydrogels the surface free energy is strongly correlated to the rate of cellular attachment. It is generally found that highenergy surfaces promote rapid cellular adhesion and spreading, whereas low energy surfaces do not favor such behavior.
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Since the primary adsorptives in culture media are extracellular proteins [11,12] the reasons for this discrimination are not obvious. It is possible the adsorption of nonattachment proteins such as albumin to hydrophobic substrates is driven by the reduction in liquid–surface interface energy by the displacement of water molecules from the surface [13]. Although the mechanism of cellular adhesion is poorly understood, the marked discrimination between surfaces of differing wettability may be used to regulate the attachment of cells at an interface [14,15]. In recent years, techniques have been developed to produce surfaces with well-defined chemical heterogeneity, which are suitable for this purpose. Surfaces containing regions of high surface energy within a low surface energy matrix are known to adsorb cells in a spatially selective manner. Such spatial control has been successfully demonstrated using patterned self-assembled monolayers (SAMs), typically using a photolithographic or a ‘rubber stamp’ technique [16] and the spatial resolution of this method is sufficient to produce patterns of subcellular dimensions. It has been shown that high resolution patterns of this type can be employed to modify the size and shape of attached cells which influences cell behavior [17–23]. These studies demonstrate the possibility of directing cell growth and behavior through the use of advanced surface engineering techniques. There are many possible applications of devices employing this technology in the study of cell behavior, organization and cellular interactions, as well as in the construction of novel biomedical devices. The major drawbacks of SAMs are their labor-intensive preparation and susceptibility to oxidation [24]. This paper outlines a simple, one-step procedure to produce a well-defined, chemically heterogeneous surface with a subcellular resolution using plasma polymerization. Plasma polymerization for patterning is advantageous, as it is applicable to a wide range of substrates with little or no pretreatment. Additionally a wide range of chemical functionalities may be deposited, albeit with much less specificity than is achievable using SAMs. However, careful control of the plasma conditions enables the production of surfaces with well defined chemistry and wettability. This work demonstrates that plasma polymers may be patterned with subcellular dimensions and highlights some of the factors that may be of importance in achieving high spatial resolution using this approach.
2. Experimental 2.1. Plasma polymerization The internal surfaces of 55 mm diameter polystyrene (PS) dishes (Nunc, UK) were exposed to an acetone plasma in home-built plasma chamber described in detail elsewhere [25]. The RF power at 13.56 MHz was coupled to the chamber via a manual impedance matching network (Coax-
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ial Power Systems Ltd., UK) using an externally wound copper coil. A Pirani gauge and digital controller (Terranova, USA) monitored pressures before and after each deposition. A base pressure of typically 1 × 10−3 mbar was achieved using a rotary vacuum pump (Welch Thomas Vacuum Model 8917C, USA) fitted with a LN2 cooled trap (Kurt J. Lesker, UK). The leak rate was measured before each plasma deposition process and at all times was 0.02 cm3 (STP) min−1 . Analytical grade acetone (Sigma Chemical, Germany) was degassed through several freeze– pump–thaw cycles and introduced into the plasma reactor through a fine control leak valve (LV10K, BOC Edwards, UK). An RF power of 10 W and a constant flow rate of ∼15, ∼44 or ∼56 cm3 (STP) min−1 was used in all experiments with a range of deposition times from 10 to 600 s. Samples for use in cell culture experiments were washed with HPLC grade water (Millipore, resistivity 18 M cm) with gentle agitation for 60 min and dried under aseptic conditions for 120 min. The production of chemically heterogeneous surfaces was achieved using a simple masking technique [26] whereby a copper transmission electron microscope (TEM) grid was placed on the surface of the PS dish and exposed to the plasma. After treatment was complete the TEM grid was gently removed from the surface to reveal the chemically patterned surface. 2.2. Cell culture Human fibroblast cells (1BR.3N) were grown in 75 cm2 / canted neck tissue culture polystyrene, TCPS flasks (Nunc) and incubated at 37 ◦ C under a 5% CO2 atmosphere. Culture media was minimum essential medium (MEM) (Labtech International, Ringmer, UK) with HEPES modification containing 25 mM HEPES, 10% (v/v) Foetal Calf Serum (Labtech International), 1 unit/ml penicillin (Labtech International), 100 µg/ml streptomycin solution (Labtech International), 100 µg/ml nonessential amino acids (Labtech International) and 2 mM L-glutamine (Lancaster Synthesis, UK). Prior to harvesting, cells were rinsed in 2 × 10 ml phosphate buffered saline (PBS) and harvested with 2 ml of Trypsin–EDTA solution (Labtech International) for a maximum of 5 min at 37 ◦ C. The trypsin was deactivated by the addition of 20 ml media culture. After centrifugation for 3 min at 340g, the pelleted cells were gently resuspended in fresh media. For the cell attachment profile experiments, a suspension of cells, ∼10,000 cells/ml, was added to each acetone plasma treated PS dish and untreated control dish. The dishes were left undisturbed at 37 ◦ C in a 5% CO2 atmosphere between observations. To determine their attachment profile the cells were observed at 10× magnification at 24, 48 and 72 h and counted using an overlaid grid with dimensions calibrated using a graticule observed at the same magnification. Chemically patterned surfaces were also observed using the above apparatus after exposure to an atmosphere of ∼95% humidity.
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2.3. X-ray photoelectron spectroscopy (XPS) The surface chemical composition of acetone plasma treated dishes was studied using a Kratos Axis HSi 5 channel imaging X-ray photoelectron spectrometer using monochromated AlKα radiation (1486.6 eV) operated at 150 W in a residual vacuum of <4 × 10−9 mbar with the analyzer in fixed transmission mode. Charge neutralization was used for all samples with the standard operating conditions for insulator surfaces: −2.3 V bias voltage, 1.0 V filament voltage and 1.9 A filament current. All spectra were acquired in hybrid mode, i.e., using both the electrostatic and the magnetic lenses. Elemental surface compositions were calculated from survey spectra measured at a pass energy of 80 eV with the error of the quantification estimated at ±10%, deduced from the analysis of a PTFE standard.
Fig. 1. Surface oxygen obtained when varying the W/FM parameter of an acetone plasma treatment. Error bars represent 1 SEM. The line shown is a guide to the eye.
2.4. Atomic force microscopy (AFM) The topography of acetone plasma treated surfaces was studied using a Digital Instruments (DI) Nanoscope IIIa SPM under ambient conditions. The surfaces were imaged in contact mode using a silicon nitride tip. Image analysis was carried out using DI version 4.23r6 software. Surface roughness (RMS) was calculated from 3 different 20 ×20 µm areas on each treated surface. 2.5. Contact angle (CA) The wettability of washed acetone plasma treated surfaces was assessed by photographing a static 20 µl droplet of doubly distilled water on the surface with a digital camera. The contact angle of the droplet on the surface was measured directly from the image.
3. Results and discussion 3.1. XPS analysis of acetone plasma modified PS The composite parameter (W/FM) is used as a measure of the total energy per unit mass of gas where W, F and M are the RF power, monomer flow rate and molecular weight of the monomer respectively. The parameter W/FM can be used to characterize the plasma conditions during the modification process. Increasing W/FM results in a higher degree of fragmentation and molecular rearrangement whereas lower values of W/FM leads to a greater retention of the chemical functionality of the monomer. The surface oxygen concentration of polystyrene exposed to an acetone plasma for 60 s as a function of the W/FM parameter is shown in Fig. 1. It can be seen that the surface oxygen concentration reaches a saturation value of ∼12 at.% for a W/FM value of ∼0.8. Further increases in W/FM does not lead to increased oxygen levels. Acetone (CH3 COCH3 ) has an oxygen content of 25 at.% so it is clear that with all plasma powers used
some oxygen is lost, probably in the form of volatile species such as CO and CO2 [27]. Several studies using acetone as a monomer have shown it to produce films with very low deposition rates [28]. It should be stressed that the results shown in Fig. 1 are likely to be from films with thickness less than the analysis depth of the XPS technique. However, oxygen can also be introduced to the polystyrene surface via the quenching of free radicals produced by vacuum ultraviolet (VUV) irradiation. The variation in surface oxygen concentration shown in Fig. 1 may reflect differences in VUV intensity produced by plasmas of different flow rates. A previous study by Ertel et al. using plasma polymerized acetone found that the growth of bovine aortic endothelial cells correlated well with the carbonyl content of the films but did not correlate with the concentration of hydroxyl or carboxyl groups [29]. For this reason we used low values of W/FM to minimize monomer fragmentation and to produce films containing a high concentration of carbonyl groups. The surface oxygen concentration of the plasma deposited films with flow rates of ∼15, ∼44 and ∼56 cm3 (STP) min−1 and RF powers of 10 W are shown in Figs. 2a, 2b and 2c respectively. The acetone plasma treatments resulted in an increase in surface oxygen concentration from 0% to (a) ∼17%, (b) ∼8% and (c) ∼12%. For each flow rate there is initially a rapid increase in surface oxygen concentration followed by a more gradual increase. The results shown in Figs. 1 and 2 were obtained from unwashed surfaces. Washing with water resulted in minimal change in the surface oxygen concentration (<2 at.%) which is within the error of the XPS technique. Further information on surface chemistry changes resulting from the deposition process has been obtained by analysis of the carbon 1s peak envelope. An example of the functional group distribution of a PS surface exposed to the acetone plasma for 600 s at a flow rate of ∼15 cm3 (STP) min−1 is shown in Fig. 3. The C 1s peak can be resolved into 6 components: C–C/C–H at 285.0 eV; βC–C/C–H which is βshifted by +0.4–0.6 eV due to the juxtaposition of carbons
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Fig. 2. Surface oxygen concentration of the acetone plasma treated PS surfaces. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. Error bars represent 1 SEM. The lines shown are guides to the eye.
Fig. 3. Peak fitted C 1s XP spectrum for a polystyrene surface exposed to the acetone plasma for 600 s with a flow rate of ∼15 Sccm.
bonded to oxygen; C–OR/βC–OR at a shift of +1.6; C=O with a shift of +3.0; O–C=O groups with a shift of +4 eV and a peak due to π–π ∗ shake-up satellites with a shift of +5–8 eV [30–32]. The relative areas of these peaks as a function of treatment time are shown in Fig. 4. This shows that the oxidation process initially proceeds via the formation of C–OR and carbonyl groups up to approximately 120 s exposure. At treatment times over 120 s the concentration of C–OR and C=O groups continue to increase but at a rate less than that of the first 120 s. For a flow rate of ∼15 cm3 (STP) min−1 the concentration of O–C=O groups starts to increase after approximately 120 s, whereas for flow rates of ∼44 and ∼56 cm3 (STP) min−1 the increase begins between 180 and 300 s. The π–π ∗ functionality appears to decrease rapidly
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Fig. 4. Oxygen functional group distribution of acetone plasma treated PS as a function of exposure time. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. (C–O, 2; C=O, "; O–C=O, Q; π –π ∗ , F.) The lines shown are guides to the eye.
within the first 120 s of treatment followed by a more gradual reduction. A reduction of the intensity of this peak, which originates from the aromatic structure of polystyrene, could be due to the deposition of a plasma polymer film or a disruption of the phenyl groups. Studies of UV-ozone oxidation of polystyrene is also known to reduce the intensity of this peak which has been attributed to ring-opening reactions [33–35]. Exposure of polystyrene to short wavelength ultraviolet of the type generated in a glow discharge is known to lead to polymer chain scission with accompanied abstraction of hydrogen and the formation of a number of radical species [36]. In an oxygen-containing discharge, such as an acetone plasma, it is thermodynamically favorable for these radicals to react with gaseous species present in the reaction chamber [37]. However, there may also be a degree of oxygen incorporation due to quenching of residual free radicals on exposure to air. It is likely that a combination of these processes is responsible for the observed oxygen incorporation. 3.2. Contact angle analysis of acetone plasma modified PS The water contact angle of acetone plasma treated PS surfaces with monomer flow rates of (a) ∼15, (b) ∼44 and (c) ∼56 cm3 (STP) min−1 are shown in Fig. 5. The acetone plasma treatments resulted in a reduction in water contact angle from ∼92 to (a) ∼72, (b) ∼70 and (c) ∼77◦ . The reduction in contact angle is to be expected with the deposition of a film containing hydrophilic oxygen species.
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Fig. 5. Water contact angle of acetone plasma treated PS surfaces. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. Showing the increased wettability with acetone plasma treatment time. Error bars represent 1 SEM. The line shown is a guide to the eye.
3.3. Wettability of acetone plasma micropatterned PS Chemically heterogeneous polystyrene surfaces were prepared by exposing surfaces to the acetone plasma with selected areas masked. A 250 mesh TEM grid with a 30 µm bar width masked a PS dish that was exposed to the plasma with a flow rate of ∼15 cm3 (STP) min−1 and 10 W power for 300 s. The dish was exposed to an atmosphere of high humidity (∼95%) to visualize the chemical pattern. Fig. 6 shows the contrast between the untreated regions (area I) and those exposed to the plasma (area II). There is a larger incidence of water droplets on the unmasked, treated, areas with little adsorbed water on the more hydrophobic, untreated areas. 3.4. AFM analysis of plasma treated PS AFM measurements found no systematic variation in surface roughness for any of the flow rates used and the films were found to follow the topography of the PS substrate which has a RMS roughness of between 3 and 5 nm [38]. Deposition depths were determined by lightly scratching the acetone plasma film deposited on a silicon wafer with a scalpel blade and imaging the resulting trenches with the AFM. A similar scratching force to that used on the coated surfaces on an uncoated silicon wafer did not result in the formation of a trench. Film deposition rates obtained for flow rates of ∼15, ∼44 and ∼56 cm3 (STP) min−1 were 0.43, 0.18 and 0.47 nm min−1 respectively. The reduced deposition rate with a flow rate of 44 cm3 (STP) min−1 suggests that a higher degree of ablation or etching is occurring at this flow
Fig. 6. Optical image of micropatterned PS surface that has been exposed to an acetone plasma for 300 s then exposed to an atmosphere of ∼95% humidity for 5 s. Patterning achieved using a 250 mesh transmission electron microscope grid (100 µm pitch, 25 µm bar and 75 µm hole). I, unmasked (treated) area; II, masked (untreated) area. Scale bar = 100 µm.
rate. Etching of polymer surfaces will occur in an oxygencontaining plasma and the etch rate will increase with increasing flow rate. As flow rate increases further the etch rate will reach a maximum and begin to decrease as there is no longer sufficient RF energy to dissociate the monomer to form reactive oxygen species [39]. 3.5. Cell culture on acetone plasma modified PS surfaces The rate of attachment and spreading of human fibroblast cells (1BR.3N) on the acetone plasma treated surfaces was significantly greater than on native PS. Images obtained from regions of a chemically patterned surface show that under otherwise identical culture conditions, cells on the treated areas exhibit a flat morphology indicating relatively strong attachment and spreading, whereas the majority of those on the untreated regions were rounded, a sign indicative of poor attachment. Rinsing the surface with phosphate buffered saline (PBS) removed the majority of cells from the untreated regions which further demonstrated their poor attachment. The proliferation of 1BR.3N cells was monitored on untreated and treated PS surfaces. The fold increase was used as a measure of cell proliferation and is calculated by dividing the number of spread cells after 48 h by the number of spread cells counted after 24 h. Data points marked with an asterisk, in Fig. 7, represent a surface oxygen concentration that shows a significant difference (P < 0.05, by t-test) in fold increase observed between untreated PS (0% surface oxygen) and that of the data point. For comparison, a data point for tissue culture polystyrene is also shown in Fig. 7. Fig. 7 shows the fold increases of 1BR.3N cells grown on acetone plasma treated PS surfaces treated with monomer flow rates of ∼15, ∼44 and ∼56 cm3 (STP) min−1 . For each
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increases in surface oxygen leads to lower proliferation rates although for all treated surfaces the rate of cell growth is significantly greater than that found on untreated polystyrene. Interestingly, for each flow rate, the surface which exhibited the greatest fold increase had no XPS-detectable carboxyl (O–C=O) content, a functional group previously shown by a number of studies to support growth and proliferation of human fibroblasts [27]. This supports the hypothesis that surface wettability rather than the concentration of any particular functional group is the most important determining influence on a materials ability to support cell attachment and proliferation. It has been shown in a number of studies that plasma modified polymer surfaces can enhance the attachment of anchorage-dependent cells such as endothelial and fibroblasts via the adsorption of cell adhesion proteins such as fibronectin and vitronectin [40,41]. The adsorption of these proteins is controlled by a number of factors including: wettability, topography and functional group distribution. The functional group distribution will influence the conformation and composition of the adsorbed protein layer. Protein adsorption onto a substrate is therefore important in controlling cellular interactions with synthetic surfaces in vivo and in vitro in the presence of serum. Since protein adsorption from serum-containing media occurs rapidly the direct recognition of surface functional groups by the cells is virtually impossible and thus functional groups are believed to affect cell adhesion via interaction of the adsorbed protein layer. Therefore, differences observed in cell attachment and growth is most likely due to differences in the adsorption of proteins by the polymer surface. Cell culture surfaces (prepared by gas plasma (glow discharge) methods) are typically chemically complex and contain a variety of chemical functional groups and it is often difficult to determine the effect of any one chemical functionality on attachment and growth. Furthermore, it should be stressed that the findings presented here are for one cell type and it may be the case that different cell types will respond differently to the same modified surface. 3.6. Cellular patterning using acetone plasma modification of PS Fig. 7. Fold increase of 1BR.3N cells on acetone plasma treated surfaces relative to surface oxygen concentration as determined by XPS. Flow rates used were (a) ∼15, (b) ∼44 and (c) ∼56 Sccm. Error bars represent 1 SEM. The line shown is a guide to the eye. A data point for tissue culture polystyrene (2) has been included for comparison.
flow rate there appears to be a sharp increase in proliferation rates even at very low surface oxygen concentrations. For flow rates of 15 and 56 cm3 (STP) min−1 the maximum proliferation is found with a surface oxygen concentration of ∼5 at.% with fold increases of ∼2 and 16, respectively. For films prepared with a flow rate of 44 cm3 (STP) min−1 the maximum fold increase of ∼7 occurs at an oxygen concentration of ∼1.5 at.%. For all plasma treatments, further
To obtain patterns of cellular resolution the masking technique described above was employed to restrict surface modification to well defined areas of the PS dishes. Fig. 8 shows images of an acetone plasma patterned PS dish that has been seeded with 1BR.3N cells and incubated for (a) 72, (b) 96, (c) 120, (d) 144 and (d) 168 h. The acetone plasma treatment used was at a flow rate of 15 cm3 (STP) min−1 for 60 s using a Sjostrand TEM grid to mask selected areas. At low incubation times the pattern is not clearly defined but as the cells grow it is clear that cells have attached predominately on the unmasked (treated) areas and spread preferentially in the direction of the treated surface resulting in aligned, elongated cells. At extended incubation periods (168 h) the encroachment of cells onto the untreated areas
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(a)
(b)
(c)
(d)
(e)
Fig. 8. 1BR.3N cells grown on acetone plasma patterned PS dish after (a) 72, (b) 96, (c) 120, (d) 144 and (e) 168 h incubation. 60 s exposure, TEM grid used was a Sjostrand mesh. Scale bars = 100 µm.
occurs after the treated zones become confluent. Figs. 8d and 8e show the cells several abreast in the bars of treated surface and have remained partially aligned, presumably due to the spreading constraints on those cells close to the chemical boundary. At present the exact mechanism by which cells spread from cell-friendly oxidized areas onto neighboring hydrophobic areas is not known.
faces composed of both hydrophilic and hydrophobic zones under otherwise identical conditions. At short incubation periods the cells preferentially attached to the hydrophilic, plasma treated areas until confluence was reached. Extended exposure caused spreading of the cells to the untreated, hydrophobic areas. Since cell line and primary cells do not normally attach to untreated polystyrene this finding seems paradoxical and may reflect changes in the cellular environment which warrant more detailed study.
4. Conclusion This study has shown that it is possible to produce surfaces with controllable oxygen chemistry using an acetone plasma. The chemistry and wettability of the surfaces were studied using XPS and contact angle measurements which demonstrated that even short plasma treatments resulted in significant changes to the polystyrene surfaces. Careful control of plasma conditions: exposure time, RF power and flow rate, allowed the production of thin films (thickness <1 nm) with no carboxyl groups. Oxygen levels of up to 17 at.% were found for samples exposed with monomer flow rates of 15 cm3 (STP) min−1 . The exact mechanism of oxygen incorporation is not clear but there are several mechanisms that may be relevant for this system including film deposition, ablation, UV irradiation from the glow discharge, postplasma oxidation after exposure to air, or a combination of these. The deposition rates found with this process were very low at approximately 0.5 nm min−1 allowing the production of chemically patterned surfaces with very abrupt chemical gradients. This allowed the study of cell attachment to sur-
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