Research Article

Received: 12 June 2008 Accepted: 25 August 2008 Published online in Wiley Interscience: 12 November 2008

(www.interscience.com) DOI 10.1002/sia.2964

Simple poly(dimethylsiloxane) surface modication to control cell adhesion

Min-Hsien Wu
Poly(dimethylsiloxane) (PDMS) has a long history of exploitation in a variety of biological and medical applications. Particularly in the past decade, PDMS has attracted interest as a material for the fabrication of microuidic biochip. The control of cell adhesion on a PDMS surface is important in many microuidic applications such as cell culture or cell-based chemicals/drug testing. Unlike many complicated approaches, this study reports simple methods of PDMS surface modication to effectively inhibit or conversely enhance cell adhesion on a PDMS surface using Pluronic surfactant solution and poly-L-lysine, respectively. This research basically succeeded our prior work to further conrm the long-term capability of 3% Pluronic F68 surfactant to suppress cell adhesion on a PDMS surface over a 6-day cell culture. Microscopic observation showed that the treated PDMS surface created an unfavorable interface, where chondrocytes seemed to clump together on day 2 and 6 after chondrocyte seeding, and there was no sign of chondrocyte spreading. On the opposite side, results demonstrated that the poly-L-lysinetreated surface signicantly increased broblast adhesion by 32% in contrast to the untreated PDMS, which is comparable to the commercial cell-culture-grade microplate. However, bronectin treatment did not have such an effect. All these fundamental information is found useful for any PDMS-related application. Copyright c 2008 John Wiley & Sons, Ltd. Keywords: poly(dimethylsiloxane); PDMS; cell adhesion; surface modication; protein adsorption

Introduction
The performance of biomaterials depends largely on protein adsorption and subsequently induced responses such as cell adhesion and thrombus.[1] The control of interfacial phenomena by adequate surface modication approaches has become more and more important. Poly(dimethylsiloxane) (PDMS) has a long history of exploitation in a variety of biological and medical applications[2] because of its biocompatibility[3,4] and low toxicity.[5] Particularly in recent years, PDMS has attracted interest in microuidic use due to its inherent properties of low cost and ease of fabrication, especially for rapid prototyping of microuidic structures.[6] However, a number of disadvantages of PDMS compared with glass or quartz hinder its wide use as a substrate for microuidic applications. PDMS is a hydrophobic material (contact angle: 105 ), which is difcult to wet with aqueous solution and prone to adsorb proteins or small hydrophobic molecules. Anderson et al.[7] have revealed that PDMS adsorbed plasma proteins very rapidly and the composition of proteins adsorbed varied dynamically due to the Vroman effect, a dynamic protein adsorption process where proteins compete for adsorbing on a surface. Thus, any contact of protein-containing solution with the PDMS surface in microuidic applications will be problematic due to the inconsistency of protein composition or the loss of target or biologically important proteins due to the nonspecic protein adsorption. For instance, the application of microuidic system for cell culture[8 10] or cell-based analysis,[11] where the culture medium is normally supplemented with serum or growth factors if necessary, is very problematic. In addition, protein adsorption and consequently induced cell adhesion will cause the problem of channel clogging which, in turn, affects the ow in a microuidic system. It is widely accepted that the deterrence to cell adhesion is largely due to the resistance of protein adsorption onto a surface.[12,13] Various approaches have been developed over the

years to minimize protein adsorption and the following cell adhesion on PDMS surfaces, each having their own specic strengths and shortcomings in this area. It is commonly believed that hydrophilic surfaces have lower levels of protein adsorption than that of hydrophobic surfaces. Modications of a PDMS surface to render it hydrophilic have been realized by means of oxygen plasma[14,15] or UV/ozone[16] treatments. Modication of PDMS surface with oxygen plasma has become a popular tool in the microuidic application for increasing hydrophilicity and for permanent bonding of PDMS.[15] However, not only will plasmatreated PDMS undergo hydrophobic recovery after several hours in the absence of electrical activity[17] due to the migration of low-molar-mass PDMS species from the bulk to the surface,[18] but it will also reduce PDMS oxygen permeability coefcients by 4080%.[19] These cause instability in handling and hinder its utilization in cell culture-related areas, respectively. Moreover, oxygen plasma or UV/ozone treatment of PDMS requires the use of specialized equipment that is usually not available in many laboratories. Alternatively, surface modication with poly(ethylene oxide) (PEO), a water soluble, nontoxic, and nonimmunogenic polymer, has been widely used to provide hydrophilic coatings for improving biocompatibility of biomaterials and this is largely due to its ability to inhibit protein[20] and cell attachment.[21] The mechanisms contributing to this capability have been extensively investigated[22 25] and can generally be summarized as: (i) large excluded volume, (ii) the congurational entropic repulsion,

Correspondence to: Min-Hsien Wu, Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan. E-mail: mhwu@mail.cgu.edu.tw Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan

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M.-H. Wu
110 100 90 Normalized percentage (%) 80 70 60 50 40 30 20 10 0 native PDMS 10% F127 3% F127 1% F127 10% F68 3% F68 5% 1% 0.05% 0.01% Tween 20 Tween 20 Tween 20 Tween 20

Figure 1. Comparison of broblast adhesion on the native and (surfactants) treated PDMS surfaces. The PDMS surfaces were treated with Pluronic F127 [at 10, 3, 1% (w/v)], Pluronic F68 (at 10 and 3%) and Tween 20 (at 5, 1, 0.05, 0.01%) solutions. Attached cells were evaluated after 32 1 h cell culture by MTT assay and the data were normalized to that of the untreated PDMS surface. Data are given as mean standard deviation (n > 3). p < 0.05, p < 0.01.

(iii) the coverage of PEO polymer on the surface, and (iv) its low interfacial energy. The incorporation of PEO polymer onto a surface can be achieved through physical adsorption,[26] covalent coupling,[27] and synthetic procedures.[28] Physical adsorption provides a simpler route than the latter two. However, physically adsorbed PEO polymers can be replaced by macromolecules with higher afnity for the interface, which gives a surface with unstable properties. Another physical approach to produce a PEO surface is to use PEO-terminated triblock polymers, such as Pluronic, which is able to form a more stable adsorbed layer with the central hydrophobic poly(propylene oxide) (PPO) block stabilizing the molecule to a hydrophobic surface, allowing the hydrophilic PEO chains to extend out into the aqueous solution. Researches have shown that Pluronic surfactants can considerably reduce protein adsorption[25,29] and cell adhesion.[21,25,30] In addition to Pluronic family, the other surfactant candidate, Tween 20 (polyoxyethilene sorbitan monolaurate), has been proven to resist protein adsorption.[31] Similar to the Pluronic surfactants, a Tween 20 coating can be simply prepared by physical adsorption on to a surface. Besides, it has also been noted that the Tween 20 coating on a hydrophobic surface can more effectively reduce albumin adsorption (by 90%) than on a hydrophilic surface (by 15%),[31] which makes it a suitable candidate for PDMS surface modication. The abilities of Pluronic or Tween 20 surfactants to prevent protein adsorption and the consequent cell adhesion have been proved partly. However, most previous studies have focused on single or binary protein models rather than on the real environment in which the substrate will be employed. Therefore the evaluation of the effectiveness of these surfactants to inhibit protein adsorption from serum-supplemented culture medium and the subsequently induced cell adhesion is practically important. In our previous study,[32] the capabilities of Pluronic F127 and F68 and, Tween 20 surfactants, to prevent serum protein adsorption and broblast adhesion on PDMS surfaces were evaluated at various concentrations using X-ray photoelectron spectroscopy (XPS) and an MTT cell viability assay, respectively. Briey, the

results demonstrated that Pluronic surfactants (F127 and F68) were more effective than Tween 20 surfactant in both the capabilities and, within the conditions investigated, minimum protein adsorption and cell adhesion occurred at a concentration ranges slightly lower than the critical micelle concentration (CMC). Overall, with an experimental condition of 32 h, minimum serum protein adsorption and broblast adhesion were achieved using 3% Pluronic F68, which was able to reduce serum protein adsorption and broblast adhesion (Fig. 1) (replotted from our previous study[32] with a further statistic analysis) by 84.6 and 94.6%, respectively over the untreated PDMS surface. However, the long-term capability of Pluronic surfactant to deter cell adsorption is unknown. Besides, another unexplored issue is that if the treated PDMS surface has the same tendency to suppress the adhesion of other cell species. To tackle these issues, this research further investigated the long-term capability of 3% Pluronic F68 to prohibit chondrocyte adhesion on a PDMS surface directly by microscopic observation. In contrast to the PDMS surface treatments to prevent nonspecic serum protein adsorption and hence unwanted cell adhesion, the enhancement of cell adhesion on a PDMS surface is required for many applications such as microuidic-based monolayer cell culture[33] or cell-based assay/testing.[34] Cell substrate adhesion is a multistep process including initial cell contact to the substrate, attachment, spreading, and growth. It is recognized that the adhesion and proliferation of different types of cells on biomaterials depend on many surface characteristics, such as surface charge,[35] wettability (hydrophobicity/hydrophilicity),[36] chemistry,[36] microstructure,[37] roughness,[38] and mechanical properties. In standard monolayer cell culture processes, bronectin and poly-L-lysine treatments are the two commonly used approaches to enhance the cell adhesion on a surface. In this study, the effectiveness of these two treatments on the enhancement of cell adhesion on a PDMS surface was explored by quantifying the viable cells on the treated PDMS surface using MTT assay. As a whole, based on our prior work,[32] this study further revealed the long-term ability of 3% Pluronic F68 to suppress

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Poly(dimethylsiloxane) surface modication cell adhesion on a PDMS surface over a 6-day cell culture. The treated PDMS surface created an unfavorable interface, where chondrocytes seemed to clump together on day 2 and 6 after chondrocyte seeding and there was no sign of chondrocyte spreading. On the opposite side, results showed that the poly-Llysine-treated surface signicantly increased broblast adhesion by 32% over the native PDMS surface, which is comparable to the cell-culture-grade microplate. However bronectin did not have such an effect. This nding was further conrmed by long-term microscopic observation of the chondrocyte adhesion (another cell species investigated) on the poly-L-lysine-treated PDMS surface. The result demonstrated that the treated PDMS surfaces appeared favorable for chondrocyte attachment and spreading, and were comparable to the commercial multiwell microplate. Basically, this research not only succeeded our prior work to conrm the longterm capability of 3% Pluronic F68 to inhibit cell adhesion but also, conversely, provided a simple and workable route to enhance cell adhesion on a PDMS surface. All these fundamental information is found useful for any PDMS-related application. pH 7.5) solutions were aseptically added into PDMS slab-loaded well and incubated at room temperature for 1 h. After surface treatment, the treated PDMS slabs were rinsed three times with PBS solution and followed by adding 0.1 ml of prepared primary broblast cell suspension to each well with a treated or untreated PDMS slab, and placed in an incubator at 37 C for up to 32 h. For comparison purpose, standard cell-culture-grade multiwell microplate was used as a control. After incubation, the MTT assay was used to quantitatively evaluate the attached broblasts. Briey, the solution in each well was discarded and the PDMS slab was then rinsed twice with PBS. Then 0.1 ml of MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reagent (0.5 mg/ml MTT prepared in nonfetal bovine serum-supplemented DMEM) (MTT assay kit) was added to each well, followed by incubation in an incubator at 37 C for 3 h. A quantity of 0.1 ml of MTT solvent (0.1 N HCl in anhydrous isopropanol) was subsequently added to each well and mixed thoroughly by pipetting to dissolve the purple crystals formed. The absorbance of the solution was then measured using a microplate reader (Genios, Tecan Ltd, UK) at 595 and 690 nm for the tested samples and blank, respectively. The absorbance data were normalized to the untreated sample. Besides, to further explore the long-term effectiveness of the bronectin and poly-L-lysine treatments on the enhancement of cell adhesion on PDMS surfaces, microscopic observation with the same experimental procedures, described previously but for different cell species (chondrocytes), was carried out. Statistical analysis Data are presented as the mean the standard deviation of the mean of at least three separate experiments (n 3). In each experiment, each condition tested was carried out in triplicate. Signicant differences were determined using Students unpaired t-test.

Materials and Methods

Preparation of PDMS slabs PDMS was prepared by thoroughly mixing the PDMS prepolymer and the curing agent (Sylgard 184, Dow Corning, USA) in a ratio of 10 : 1 by weight. The polymer was then de-aerated under vacuum to remove all air bubbles generated during mixing. The mixture (15 g PDMS prepolymer and 1.5 g curing agent) was then poured onto a clean 3-in. silicon wafer and cured at 100 C for 10 min. The cured PDMS slab was approximately 1-mm thick. A circular (diameter: 9 mm) PDMS slab was obtained using a borer. Circular PDMS slabs were then autoclaved to sterilize. Long-term effectiveness of Pluronic F68 to prevent cell adhesion on a PDMS surface The effectiveness of Pluronic F68 surfactant treatment to prevent serum protein adsorption and the consequent cell adhesion on a PDMS surface was revealed in our previous study.[32] On the basis of this, this research further explored the long-term effectiveness of 3% (w/v) of Pluronic F68 surfactant treatment to deter chondrocyte adhesion. Briey, 2 ml of 3% Pluronic F68 solution (unless otherwise stated, all chemicals were purchased from SigmaAldrich Company Limited, Taiwan) was loaded into each well in a sterile 24-well microplate. One sterilized PDMS slab was placed in one well with at side (the side in contact with the Si wafer during molding) down onto the meniscus of the surfactant solution ensuring no air bubbles were trapped at the interface. The Pluronic F68 surfactant was adsorbed at 20 C for 24 h to ensure maximum coverage and the treated PDMS slabs were then rinsed twice with phosphate-buffered saline (PBS). After treatment, 0.15 ml of primary chondrocyte suspension of cell density of 1 105 cells ml1 was loaded into each well of a 48well microplate with a treated PDMS slab attached on the surface and subsequently placed in an incubator. Microscopic observation was carried out after 2 and 6 days of incubation using an inverted microscope and appended camera. Evaluation of bronectin and poly-L-lysine treatments to enhance cell adhesion on PDMS surfaces A quantity of 0.15 ml of sterile poly-L-lysine [0.01% (w/v), MW: 70 000150 000] and bronectin (0.01%, in 0.5 M NaCl, 0.05 M Tris:

Results and Discussion

Long-term effectiveness of Pluronic F68 to prevent cell adhesion on a PDMS surface On the basis of the previous results, the deterrence to cell adhesion is strongly correlated with the resistance of protein adsorption onto a surface.[32] Within the conditions explored, our prior work revealed that the optimum condition for preventing serum protein adsorption and the following broblast attachment (Fig. 1) on PDMS surface was Pluronic F68 treatment at a concentration of 3%, showing the reduction level of 84.6 and 94.6%, respectively, over the untreated surface. However, this study was a 32-h observation. In many cases, the long-term stability to prohibit cell adhesion on a surface is crucial, for example, microuidic-based cell-culture system for various extended applications,[39,40] where cell patterning is required on a specic zone of a PDMS surface. As mentioned earlier, the Pluronic F68 treatment adopted in this study is virtually by means of physical adsorption of PEO-terminated triblock polymers on a PDMS surface. It may mean that these polymers can be risky to desorb due to the replacement of higher afnity macromolecules existing in the serum-containing cellculture medium. To test the long-term capability of Pluronic F68 to deter cell adhesion, chondrocyte attachment and spreading on the PDMS surfaces modied with Pluronic F68 at a concentration of 3% were observed microscopically after 2 and 6 days following

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M.-H. Wu multiwell microplate surface (Fig. 2(c)) showing that chondrocytes started spreading on day 2 and became fully spreading on day 6. Fibronectin and poly-L-lysine treatments to enhance cell adhesion on PDMS surfaces
100 microns

2 days (a) 3% Pluronic

6 days F68 treated surface

(b) Native PDMS surface

(c) Multi-well microplate

Surface treatments intended to enhance cell adhesion on PDMS surface were evaluated by comparing the total viable cells on the surfaces using the MTT assay. The PDMS surfaces were treated with poly-L-lysine [0.01%(w/v)] and bronectin [0.01%(w/v)] for 1 h. Fibroblasts were seeded and cultured in 10% serum-containing medium for up to 32 h. Figure 3 showed that the broblast adhesion on a native PDMS surface was about 32% signicantly (p < 0.01) lower than that of cell-culture-grade microplates and this outcome was further justied by the long-term microscopic observation of chondrocyte adhesion on both the surfaces (Fig. 2(b) and (c)). This nding may be attributed to the difference in the wettability of surfaces tested. Webb et al.[35] has demonstrated that serum-treated hydrophilic surfaces support signicantly greater cell attachment relative to hydrophobic surfaces. Furthermore, some previous studies have also conrmed that native PDMS is not a supportive material for the cell adhesion.[36] In contrast to the untreated PDMS, the results (Fig. 3) revealed that the poly-L-lysine-treated surface signicantly (p < 0.01) increased broblast adhesion by 32%, which is comparable to the cell-culture-grade microplate. Again, this outcome was further conrmed by long-term microscopic observation of the chondrocyte adhesion on the poly-L-lysine-treated PDMS surface (Fig. 2(d)), showing that the treated PDMS surfaces appeared attractive for chondrocyte attachment and spreading, and were comparable to the microplate (Fig. 2(c)) within the experimental conditions investigated. All vertebrate cells possess unevenly distributed negative surface charges and the presence of electric charges on a substratum surface has also been shown to play a role in the cell adhesion process.[41] For poly-L-lysine treatment, the underlying principle is that the poly-cationic poly- Llysine molecule adsorbs strongly to various solid surfaces, leaving cationic sites which combine with the anionic sites on cell surfaces. Conversely, bronectin treatment did not have such an effect (Fig. 3). Fibronectin is one of many known extracellular matrix

(d) Poly-L-lysine treated surface

Figure 2. Comparison of chondrocyte adhesion on the PDMS surfaces: (a) Pluronic F68 (3%, 24 h) treated PDMS, (b) native PDMS, (c) cell-culturegrade 48-well microplate, (d) poly-L-lysine [0.01% (w/v), 1 h] treated PDMS. Microscopic photograph was taken after 2 and 6 days of cell culture. This gure is available in colour online at www.interscience.wiley.com/journal/sia.
Normalized percentage (%)

cell seeding. In this study, the surfaces of native PDMS and commercial cell-culture-grade multiwell microplate were controls, and a different cell species (chondrocytes) was demonstrated. Figure 2(a) displays that chondrocytes seemed to clump together on day 2 and 6 after chondrocyte seeding and there was no sign of chondrocyte spreading, exhibiting differently from the untreated PDMS surface (Fig. 2(b)), where, although cells tended to aggregate on day 2, there were some adhesion and spreading on day 6. All these ndings were in sharp contrast with the cell-culture-grade

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

microplate

native PDMS

Polylysine treated

Fibrinectin treated

Figure 3. Comparison of broblast adhesion on the native and treated PDMS surfaces. The PDMS surfaces were treated with poly-L-lysine [0.01% (w/v)] and bronectin [0.01% (w/v)] solutions for 1 h. The surfaces of cell-culture-grade multiwell microplate and untreated PDMS were used as controls. Attached cells were evaluated after 32 1 h cell culture by MTT assay and the data were normalized to that of untreated PDMS surface. Data are given as mean standard deviation (n > 3). p < 0.01.

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Poly(dimethylsiloxane) surface modication proteins such as vitronectin, brinogen, collagens, laminin, and other trace proteins, mediating the specic interaction of cells to surfaces via cell integrin receptors. The role of bronectin to enhance cell adhesion has been demonstrated by Webb et al.,[42] in which interference from competition by serum proteins has been excluded. Results showed that the attachment of broblasts on a surface was a function of the amount of immobilized bronectin. However, in serum-containing culture, Steele et al.[43] have revealed that selective depletion of serum bronectin did not have any effect on either cell attachment or spreading of broblasts, and that the adsorption of serum vitronectin, which can overcome the effect of serum components, tending to decrease cell attachment, determines the attachment of broblasts on tissue culture polystyrene. Besides, Knox[44] also revealed that the speed of cell spreading is sensitive to the level of serum in the culture medium. At a concentration of 3% or above, serum bronectin plays no role in serum-stimulated spreading, mainly due to its displacement by albumin and other plasma proteins as cell adhesion inhibiting proteins such as albumin preferentially adsorbed at higher serum concentration.[41] Apart from the fact that bronectin adsorbed may be displaced from surfaces by competing species in serum due to its low competitive activity[45] as mentioned earlier, another possible explanation may be the fact that the nature of surface can inuence the conformation of adsorbed bronectin. Grinnell and Feld[12] have revealed that although there is a higher adsorption of bronectin on a hydrophobic surface than on the hydrophilic counterpart, the adsorbed bronectin was less active on the hydrophobic substrate. Besides, Toworfe et al.[46] also demonstrated that although bronectin surface density was slightly higher on a native PDMS surface (a hydrophobic surface) than on the UV/ozone-activated PDMS surface (a hydrophilic surface), higher attachments of MC3T3E1 osteoblast-like cells were observed on the bronectinprecoated UV/ozone-activated PDMS surface. Taken together, the competition in serum protein adsorption due to the Vroman effect and the chemical properties of substrate determine the resulting density and conformation of the specic bronectin cellbinding domain on a surface, and hence the cell attachment. These may be able to explain why low broblast adhesion was found on a bronectin-conditioned PDMS surface in this study. terning in the microuidic-based cell-culture systems using PDMS as substrate. Acknowledgements The authors would like to thank nancial support from the National Science Council (NSC) in Taiwan (NSC97-2218-E-182-002-MY2).

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Conclusions
In this work, a simple PDMS surface treatment using 3% Pluronic F68 surfactant for stably deterring cell adhesion over long term in cell culture was further conrmed microscopically. Results demonstrated that the treated PDMS surface created an unfavorable interface, where chondrocytes seemed to clump together on days 2 and 6 after chondrocyte seeding and there was no sign of chondrocyte spreading. On the other hand, regarding PDMS surface modication for enhancing cell adhesion, two commonly used approaches (bronectin and poly-L-lysine) were compared. The results showed that poly-L-lysine treatment, comparable to commercial cell-culture-grade microplates, was more effective than the bronectin. As a whole, this study provides not only fundamental information about the capability of surfactant-based PDMS treatments to prevent cell adhesion but also a simple approach to effectively enhance cell attachment. All these are found particularly useful for controlling over cell adhesion or cell pat-

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