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Characterization of initial cell adhesion on charged polymer substrates in serum-containing and serum-free media Takashi Hoshiba, Chiaki Yoshikawa, and Keita Sakakibara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00233 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 17, 2018

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Characterization of initial cell adhesion on charged polymer substrates in serum-containing and serumfree media Takashi Hoshiba1, 2, 3, *, Chiaki Yoshikawa3, and Keita Sakakibara4 1

Frontier Center for Organic Materials, Yamagata University, 4-3-16 Jonan, Yonezawa,

Yamagata 992-8510 Japan 2

Innovative Flex Course for Frontier Organic Material Systems, Yamagata University, 4-3-16

Jonan, Yonezawa, Yamagata 992-8510 Japan 3

International Center for Materials Nanoarchitechtonics, National Institute for Materials Science,

1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 Japan 4

Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011 Japan

KEYWORDS Cell adhesion, charged polymer, protein adsorption, integrin

ACS Paragon Plus Environment

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ABSTRACT

Charged substrates are expected to promote cell adhesion via electrostatic interaction, but it remains unclear how cells adhere to these substrates. Here, initial cell adhesion (< 30 min) was re-examined on charged substrates in serum-containing and serum-free media to distinguish among various cell adhesion mechanisms (i.e., electrostatic interaction, hydrophobic interaction, and biological interaction). Cationic and anionic methacrylate copolymers were coated on nonionic non-tissue culture-treated polystyrene to create charged substrates. Cells adhered similarly on cationic, anionic, and nonionic substrates in serum-free medium via integrinindependent mechanisms, but their adhesion forces differed (anionic > cationic > nonionic substrates), indicating that cell adhesion is not mediated solely by the cells’ negative charge. In serum-containing medium, the cells adhered minimally on anionic and nonionic substrates, but they adhered abundantly on cationic substrates via both integrin-dependent and -independent mechanisms. These results suggest that neither electrostatic force nor protein adsorption is accountable for cell adhesion. Conclusively, the observed phenomena revealed a gap in the generally accepted understanding of cell adhesion mechanisms on charged polymeric substrates. A re-analysis of their mechanisms is necessary.


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1. Introduction Cell adhesion to a substrate is key for tissue engineering and regenerative medicine [1, 2]. Many properties are known to regulate cell adhesion to substrates with different surface properties, including hydrophilicity [3, 4], chemical structure [4, 5], molecular mobility [6, 7], water structure [8, 9], and electrostatic force [4, 10]. In particular, the electrostatic force strongly facilitates cell adhesion to a substrate [4, 10]; as such, charged polymer substrates are widely used for cell culture and to control cell functions [11, 12]. Many studies have attempted to elucidate the mechanism of cell adhesion on charged polymer substrates [13-18]. Chang et al. reported that the numbers of adherent NIH3T3 fibroblasts and epithelial cells (HEK293T embryonic kidney cells and HepG2 hepatocarcinoma cells) increased according to the increase of surface potential [13, 14]. Webb et al. reported that substrates with a positively charged functional group promote NIH3T3 fibroblast adhesion [15]. Lee et al. also reported that substrates with a positively charged functional group promote Chinese hamster ovary (CHO) cell adhesion [16]. It is generally believed that positively charged substrates promote cell adhesion via electrostatic interaction with the negatively charged cell membrane [19]. However, all of the aforementioned studies were performed in serum-containing media. In serum-containing media, serum proteins can be adsorbed on the substrate, raising the possibility that cells adhere via their interaction with the adsorbed serum proteins rather than with the substrate itself [20. 21]. Thus, the direct effects of electrostatic interaction on cell adhesion have not been proven. Understanding the role of electrostatic interaction in cell adhesion thus requires the examination of cell adhesion on a charged substrate in serum-free medium in which few or no proteins adsorb on the substrate.


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Moreover, tissue culture polystyrene (TCPS), a polystyrene substrate treated with plasma to make its surface negatively charged, has been used widely for cell culture. If cell adhesion is promoted by positively charged substrates, adhesion should be suppressed on negatively charged substrates such as TCPS by a repulsion force. However, cells can adhere on TCPS, even in serum-free medium [12, 22-25]. This phenomenon reveals a gap in our general understanding of the role of the electrostatic force in cell adhesion. We must therefore re-examine the effect of electrostatic force on cells in serum-free media to understand its contribution to cell adhesion. In addition to the effect of serum proteins on cell adhesion, the incubation time is critical for analysis of the phenomenon. The cells can adhere on the substrates via various mechanisms. The adhesion mechanism can be altered to the interaction between adsorbed proteins and integrins, receptors on the cell membrane, after a long period of incubation, which confounds analysis of the cell adhesion mechanism [26, 27]. Cell adhesion has been assessed after a long-term period (3 h or longer) in previous research; therefore, it is possible to misunderstand the mechanisms. Thus, adhesion mechanisms must be assessed over a shorter period. Previous studies have been conducted under experimental conditions that complicate the analysis of cell adhesion mechanisms: i) serum proteins are present and ii) the experimental period is too long to assess the interaction of cells with charged polymer substrates. We therefore propose to distinguish among the factors that influence cell adhesion on charged substrates and to elucidate the mechanism of cell adhesion under less problematic experimental conditions. In this study, we used charged polymers to create charged substrates because polymers have been widely and easily used for tissue engineering and regenerative medicine [28]. An anionic or cationic polymer was used to coat a nonionic polymer substrate, and adhesion was assessed on


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the prepared polymer-coated substrates to clarify the effects of electrostatic and hydrophobic interactions on cell adhesion (Figure 1A). Furthermore, cell adhesion was assessed in both serum-containing and serum-free media to distinguish biological interactions (i.e., adhesion to adsorbed proteins via integrins) from electrostatic and hydrophobic interactions by eliminating serum protein adsorption (Figure 1B). For the evaluation of cell adhesion mechanisms, integrin dependency and cell adhesion forces were assessed via an inhibitory cell adhesion assay using ethylenediamine tetra-acetic acid (EDTA) [29] and single-cell force spectroscopy (SCFS) [30]. Finally, we demonstrate that cells adhere via both positive and negative charges, emphasizing that the mechanisms of cell adhesion on charged substrates must be reconsidered.

2. Materials and methods 2.1. Synthesis and characterization of copolymers It is generally performed to prepare the copolymers of methyl methacrylate (MMA) with hydrophilic monomers with random copolymerization [31, 32]. In addition, it is very general method to immobilize these copolymers on the substrate surface through the hydrophobic interaction of hydrophobic moiety for prepare hydrophilic surface [33, 34]. Thus, we synthesized the MMA copolymers with methacrylic acid (MA) and (2-dimethylaminoethyl)methacrylate (DMAEMA) as coating polymers with negative and positive charges, respectively. MA (Wako, Osaka, Japan) and DMAEMA (Wako) were purified by passage through an aluminum column. MMA (Wako) was purified by distillation. 2,2'-Azobisisobutyronitrile (AIBN) (Wako) was purified by re-precipitation with ethanol.


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2.1.1. Preparation of P(MA73-co-MMA27) An N,N-dimethylformamide (DMF, Wako) solution of MA (6.45 g, 750 mM), MMA (2.5 g, 250 mM), and AIBN (41.0 mg, 2.5 mM) was purged with argon gas and then heated at 70°C for 24 h. After polymerization, an aliquot of the solution was diluted with DMF containing LiCl (10 mM) and analyzed by gel permeation chromatography (GPC) to calculate the molecular weight as described below. The weight-averaged molecular weight (Mw) was 2.4 × 104, and the polydispersity index (Mw/Mn) was 2.84. The copolymer was purified by re-precipitation using diethyl ether. The molar ratio (mol%) of polyMA to polyMMA in the polymer was determined to be 73:27 by nuclear magnetic resonance (1H NMR) (ECS-400) (JEOL, Tokyo, Japan) in dmethanol or deuterium oxide. The chemical structure of the synthesized polymer is shown in Figure 2A. 2.1.2. P(DMAEMA78-co-MMA22). A DMF solution of DMAEMA (11.8 g, 750 mM), MMA (2.5 g, 250 mM), and AIBN (41.0 mg, 2.5 mM) was purged with argon gas and then heated at 70°C for 24 h. After polymerization, the solution was analyzed by GPC to calculate its molecular weight as: Mw = 2.1 × 104 and Mw/Mn = 3.18. The copolymer was purified by re-precipitation using water. 1H NMR confirmed the molar ratio of polyDMAEMA to polyMMA in the copolymer to be 78:22. The chemical structure of the synthesized polymer is shown in Figure 2B.

2.1.3. GPC measurement


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GPC analysis of the copolymers was performed using a Tosoh CCP&8020-series high-speed liquid chromatograph (Tokyo, Japan) equipped with two Shodex gel columns, LF804 (300 × 80 mm; bead size = 6 µm; pore size = 20-3000 Å) (Tokyo). DMF containing 10 mM LiCl was used as the eluent at a flow rate of 0.8 mL/min (40°C). The column system was calibrated with poly(methyl methacrylate) (PMMA) standards (Shodex).

2.2. Preparation and characterization of polymer-coated substrates 2.2.1. Polymer coating Each polymer was dissolved in methanol (Wako) at a concentration of 1 wt% and coated on nontissue culture-treated polystyrene (NT-PSt) plates (IWAKI, Tokyo, Japan) by adding 12 µL/cm2 of the solution to each NT-PSt plate. The samples were air-dried for at least 3 days. The water contact angles of the prepared substrates were measured using the sessile drop method (2-µL water droplets) with a contact angle meter (G-1-1000, Erma Inc., Tokyo, Japan) The water contact angles of the substrates coated with P(MA73-co-MMA27) and P(DMAEMA78-coMMA22) were 48.9 ± 7.3° and 53.1 ± 2.5°, respectively. The contact angle of bare NT-PSt was 83.2 ± 0.5°.

2.2.2. Zeta potential measurement The zeta potential of the surface of the polymer-coated substrates (37×16 mm) was estimated with the zeta potential and particle size analyzer ELS-Z2 (Otsuka Electronics Co., Osaka, Japan). The monitor particles (polystyrene latex coated with hydroxypropyl cellulose of approximately


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500-nm diameter; EL-9001) were suspended in an aqueous NaCl solution (10 mM), and laser Doppler electrophoresis measurements were carried out according to manufacturer’s instructions with the solid sample cell unit (Otsuka) clamping the polymer-coated substrates at 25°C.

2.3. Evaluation of protein adsorption behavior Protein adsorption behavior was evaluated with an enzyme-linked immunosorbent assay (ELISA). Polymer-coated substrates and bare NT-PSt substrate were pre-incubated in phosphate buffered saline (PBS) for 1 h at 37°C. After pre-incubation, the substrates were incubated for 1 h in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (1:1) (DMEM/F-12) (Gibco, Carlsbad, CA) with 10% of either fetal bovine serum (FBS, Equitech-Bio, Kerrville, TX) or human serum (HS, Sigma, St Louis, MO). For the measurement of total adsorbed protein, the substrates were incubated in a solution containing 5% sodium dodecyl sulfate (SDS) and 0.1 N NaOH for 60 min at room temperature to extract the adsorbed proteins. The extracted proteins were assessed with a microBCA assay (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. The amount of protein was calculated using the albumin standard curve. To evaluate each protein’s adsorption behavior, the substrates incubated in serum-containing medium were reacted with primary antibody against albumin (Immunology Consultant Laboratory, Portland, OR), vitronectin (Proteintech, Rosemont, IL), fibronectin (Beckton Dickinson, Franklin Lakes, NJ), and conformation-specific fibronectin (HFN7.1, Abcam, Cambridge, United Kingdom) [7] for 2 h at room temperature. After the reaction, the substrates were reacted with the corresponding secondary antibody conjugated with peroxidase for 1 h at


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room temperature. To compare the amount of reacted antibodies, the substrates were incubated with 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) substrate (Roche Diagnostics). The absorbance was measured at a wavelength of 405 nm with a MultiskanGo (Thermo Fisher Scientific, Waltham, MA). The theoretical isoelectric points of albumin, vitronectin, and fibronectin were calculated from amino acid sequences on the website [35].

2.4. Cell culture experiments 2.4.1. Cells The human fibrosarcoma cell line HT-1080 and the human cervix epithelioid carcinoma cell line HeLa were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan). HT-1080 cells and HeLa cells were used as models of stromal and epithelial cells, respectively. The cells were maintained in DMEM/F-12 medium containing 10% FBS in TCPS flasks (IWAKI). The cells were harvested by treatment with trypsin/EDTA (Gibco) and were used for further experiments.

2.4.2. Cell adhesion assay The polymer-coated substrates were immersed in DMEM/F-12 medium with or without 10% FBS for 1 h at 37°C prior to the assay. The cells were seeded onto the substrates at a density of 3 × 104 cells/cm2. The cells were allowed to adhere to the substrate for 5, 10, or 30 min or 24 h at 37°C in DMEM/F-12 medium with or without 10% FBS. The non-adherent cells were removed


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by washing the plate once with PBS, and the adherent cells were fixed with 0.1% glutaraldehyde overnight at room temperature. Adherent cells were visualized by staining with crystal violet (Wako). After visualization, the adherent cells were counted in three randomly selected fields using an optical microscope. For the inhibition assay, the cells were treated with 5 mM EDTA for 10 min at 37°C prior to seeding. The cell adhesion assay was performed after incubating for 30 min in the presence or absence of EDTA.

2.4.3. Single-cell force spectroscopy (SCFS) Tipless cantilevers (Arrow-TL1-50, NanoWorld, Neuchâtel, Switzerland) were incubated in PBS containing 10 µg/mL FN and poly-L-lysine (PLL, MP Biomedicals, Solon, OH) for 30 min at room temperature to capture a single suspended cell. After incubation, the cantilevers were washed with MilliQ water twice and were dried for 1 h. The cantilevers were used within 3 days. The experiments were performed using the CellHesion 200 module (JPK Instruments AG, Berlin, Germany) installed on an inverted microscope (Olympus, Tokyo, Japan). The spring constant of the cantilever was determined before experiments by using the manufacturer’s software (JPK Instruments AG) based on the thermal noise method in air. To capture a single HT-1080 or HeLa cell on the cantilever, the cells were suspended in DMEM/F-12 medium with 10% FBS on a substrate coated with the copolymer of 2methacryloyloxyethyl phosphorylcholine and butyl methacrylate (30:70 mol%) (generously gifted by NOF, Tokyo, Japan). The cantilever was pressed against a single free cell by performing a force curve (the set-point force: 7 nN). Then, the cantilever was retracted and rested for 10 min to allow for strong cell adhesion between the cell and the cantilever.


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The cantilever-adhered cell contacted the substrate in DMEM/F-12 medium with or without 10% FBS. The cell was pressed onto the substrate for 10 sec with 2 nN of set-point force in constant force mode. Next, the cell-adhered cantilever was retracted for 50 µm at a rate of 2 µm/sec. The manufacturer’s software (JPK Instruments AG) was used to perform image processing and data analysis.

2.5. Statistical analysis All of the data are expressed as the means ± SD. The significance of the difference between two samples was determined using an unpaired Student’s t test in Microsoft Excel 2010. Statistical analyses on the differences among three or more samples were performed using R, a language and environment for statistical computing. The significance of the differences was determined using an analysis of variance (ANOVA). Tukey’s multiple comparison test was applied as a post-hoc test. Differences with P values less than 0.05 were considered statistically significant.

3. Results 3.1. Protein adsorption behavior on charged polymer substrates First, the surface zeta potential was measured to electrostatically characterize the polymer substrates. The surface zeta potentials of P(MA73-co-MMA27) and P(DMAEMA78-co-MMA22), calculated based on the electrophoretic profiles of polystyrene latex, were -33.7 ± 3.4, and 31.1 ± 1.1 mV, respectively. These values are reasonable because under neutral experimental conditions, the carboxylic acid and secondary amine moieties in P(MA73-co-MMA27) and P(DMAEMA78-


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co-MMA22) are converted to carboxylate (anion) and ammonium salt (cation), respectively. In contrast, the zeta potential of bare NT-PSt substrate was found to be -18.0 ± 0.06 mV, which will be discussed later. Cell adhesion is influenced by adsorbed proteins on the substrates from the medium [19, 20]. Thus, the amount of total adsorbed protein was assessed by the µBCA assay (Figure 3A). Few significant differences were observed among the substrates by this method. For further evaluation, we focused on specific protein components known to influence cell adhesion and measured their amounts by ELISA with specific antibodies. The amount of adsorbed albumin (which inhibits cell adhesion, Figure 3B) and vitronectin (which promotes cell adhesion, Figure 3C) on P(MA73-co-MMA27) were similar to those on bare NT-PSt. In contrast, the amounts of adsorbed albumin and vitronectin on P(MA73-co-MMA27) and bare NT-PSt were 1.6-2.7-times higher than on P(DMAEMA78-co-MMA22). In the case of fibronectin (which promotes cell adhesion), the adsorbed protein amount on P(DMAEMA78-co-MMA22) was similar to that on bare NT-PSt and was approximately 22% lower than on P(MA73-co-MMA27) (Figure 3D). Conformational changes in fibronectin have been shown to strongly influence cell adhesion [36]. Thus, conformational change was also compared among substrates by ELISA with a conformation-specific HFN7.1 antibody [7]. The amount of conformationally changed fibronectin on P(MA73-co-MMA27) and bare NT-PSt substrates was 2.1-2.2 times higher than that on P(DMAEMA78-co-MMA22) (Figure 3E). Finally, we calculated the ratio of conformationally changed fibronectin to total adsorbed fibronectin. The ratios (total fibronectin/HFN7.1) on P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt were 0.45, 0.27, and 0.53, respectively.


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3.2. Cell adhesion after long-term culture Next, cell adhesion was assessed on charged polymer substrates in serum-containing and serumfree media after 24 h in culture (Figure 4). In this study, HT-1080 and HeLa cells were used as stromal and epithelial cell models, respectively. Both HT-1080 and HeLa cells adhered similarly on P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and NT-PSt in serum-free medium. These results indicated that cells can adhere on the substrates even in serum-free medium. In contrast to the adhesion observed in serum-free medium, cell adhesion on NT-PSt was lower in serumcontaining medium than serum-free medium. In addition, adhesion on P(DMAEMA78-coMMA22) was slightly increased in serum-containing medium compared with the adhesion observed in serum-free medium. These differences in cell adhesion between serum-containing and serum-free media might be due to the adsorbed serum proteins. On the other hand, there were no obvious differences in adhesion on P(MA73-co-MMA27) between serum-containing and serum-free media. Thus, these results indicate the necessity of the confirmation of cell adhesion in both serum-containing and serum-free media.

3.3. Cell adhesion profiles on charged polymers We next examined the adhesion profiles of HT-1080 and HeLa cells on the charged polymers in DMEM/F-12 medium with or without FBS. Both HT-1080 and HeLa cells began to adhere on P(DMAEMA78-co-MMA22) within 5 min in serum-containing medium (Figure 5A and 5C). In contrast, the cells started to adhere on P(MA73-co-MMA27) after 30 min in serum-containing medium (Figure 5A and 5C). The cells hardly adhered on bare NT-PSt after 30 min in serum-


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containing medium (Figure 5A and 5C). Compared with the adhesion in serum-containing medium, both HT-1080 and HeLa cells started to adhere on P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt substrates within 5 min in serum-free medium (Figure 5B and 5D). These results indicated that cell adhesion profiles do not differ for cationic, anionic, and nonionic polymer substrates in serum-free medium; however, these profiles changed, and the differences appeared in serum-containing medium. The shapes of adhered cells remained round on P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt after 30 min (Supplemental Figure 1).

3.4. Cell adhesion mechanisms on charged polymers When the cells adhered on polymeric substrates in the medium-containing serum, integrins, which are situated on the cell membrane, facilitate interaction with the adsorbed proteins [27, 37]. Thus, the integrin dependency of adhesion on the charged polymers was examined using an inhibitory cell adhesion assay with EDTA, which inhibits integrin-dependent adhesion [29] (Figure 6). The number of adherent HT-1080 and HeLa cells decreased to baseline on P(MA73co-MMA27) in the presence of EDTA in serum-containing medium, indicating that the cells adhered on P(MA73-co-MMA27) via an integrin-dependent mechanism. However, both HT-1080 and HeLa cells still adhered on P(DMAEMA78-co-MMA22) in the presence of EDTA in serumcontaining medium, indicating that the integrin dependency of cell adhesion was lower on P(DMAEMA78-co-MMA22) than on P(MA73-co-MMA27). These results suggested that the cells adhered on P(DMAEMA78-co-MMA22) via both integrin-dependent and -independent mechanisms. The cells hardly adhered on bare NT-PSt after 30 min in serum-containing medium


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in the presence and absence of EDTA. This finding indicates that the integrin dependency differs among cationic, anionic, and nonionic polymer substrates, even in the presence of adsorbed proteins (i.e., in the medium with serum). In serum-free medium, EDTA did not completely inhibit the adhesion of both HT-1080 and HeLa cells, indicating that the cells adhered on the substrates via integrin-independent mechanisms. Finally, we measured the cell adhesion forces by single-cell force spectroscopy to further characterize cell adhesion on the charged polymer substrates (Figure 7). The adhesion forces in serum-containing medium were lower than those in serum-free medium, except for the force of HT-1080 cells on P(DMAEMA78-co-MMA22). Compared the cell adhesion force on the substrates in serum-containing medium, the cell adhesion forces of both HT-1080 and HeLa cells were as follows: P(DMAEMA78-co-MMA22) > P(MA73-co-MMA27), bare NT-PSt. The forces on P(MA73-co-MMA27) and bare NT-PSt were below 400 pN in serum-containing medium. The force on P(DMAEMA78-co-MMA22) was over 800 pN in serum-containing medium. In contrast, the adhesion forces in serum-free medium were as follows: P(MA73-co-MMA27) > P(DMAEMA78-co-MMA22) > bare NT-PSt. However, the force on bare NT-PSt was over 750 pN.

4. Discussion 4.1. Different protein adsorption behaviors on charged polymer substrates In this study, anionic P(MA73-co-MMA27) and cationic P(DMAEMA78-co-MMA22) were used to prepare substrates with negative and positive charges, respectively. We confirmed the zeta potential of the substrate surface by laser-Doppler electrophoresis measurement in 10 mM NaCl


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aqueous condition. Compared to zeta potentials measured in NaCl solution and in PBS, there are differences due to the effect of ionic strength, but the tendency is the same. In addition, it is expected that zeta potentials measured with and without serum would not be significantly different because of the lower effect on ionic strength. Thus, we compared zeta potential in NaCl aqueous solution here. Zeta potential measurements confirmed that the substrates coated with P(MA73-co-MMA27) and P(DMAEMA78-co-MMA22) possessed anionic and cationic charges, respectively. Furthermore, the NT-PSt substrate was negatively charged, consistent with a previous report [25]. However, its negative charge was smaller than that of P(MA73-co-MMA27). It is generally believed that electrostatic forces promote protein adsorption [38]. However, the amount of total adsorbed protein present was similar among anionic P(MA73-co-MMA27), cationic P(DMAEMA78-co-MMA22), and nonionic NT-PSt. This is likely due to the abundance of proteins in serum-containing medium and the lengthy incubation time (1 h), which was sufficient to reach equilibrium for protein adsorption on the substrates. To discuss whether the adsorbed proteins formed multiple layers, the amount of albumin required for multilayer formation was calculated using a model. Albumin (Mw. 67,500) is reported to adsorb in side-on form and is modeled as an ellipsoid with the dimensions of 4 nm × 4 nm × 14 nm [39]. Thus, the amount of monolayer-adsorbed albumin is calculated as approximately 230 ng/cm2. Based on this calculation, it seems that the adsorbed proteins form a multilayer on the substrates. The most superficial layer of adsorbed proteins is important for cell adhesion because only this layer interacts with cells. Thus, the adsorption amounts were evaluated by ELISA, which detects proteins on the most superficial layer. The amounts of adsorbed albumin, vitronectin, and fibronectin varied among cationic, anionic and nonionic polymer substrates (Figure 3B-3D). The theoretical isoelectric points of albumin, vitronectin, and fibronectin were calculated as 5.82,


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5.92, and 5.49, respectively. Thus, these proteins might be negatively charged in the medium. It is therefore expected that anionic P(MA73-co-MMA27) will suppress the adsorption of negatively charged proteins and that cationic P(DMAEMA78-co-MMA22) will promote the adsorption of negatively charged proteins. Although negatively charged albumin, vitronectin, and fibronectin adsorbed well on P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt, lower adsorption amounts were detected on P(DMAEMA78-co-MMA22) than on P(MA73-co-MMA27) (Figure 3B-3D). Serum contains both positively and negatively charged proteins that can be adsorbed on the substrates. In the case of P(MA73-co-MMA27), positively charged proteins might be dominantly adsorbed on the surface due to their negative charge. Additionally, negatively charged proteins, such as albumin, vitronectin, and fibronectin, can be adsorbed via positively charged proteins adsorbed on P(MA73-co-MMA27) (Supplemental Figure 2A). In contrast, negatively charged proteins might be dominantly adsorbed on P(DMAEMA78-co-MMA22) due to their positive charge. Following the adsorption of negatively charged proteins, positively charged proteins can then be adsorbed via negatively charged proteins adsorbed on P(DMAEMA78-coMMA22) (Supplemental Figure 2B). Thus, more negatively charged albumin, vitronectin, and fibronectin were detected on P(MA73-co-MMA27) than on P(DMAEMA78-co-MMA22). Similarly, more conformationally changed fibronectin might be detected in the most superficial layer of proteins adsorbed on P(MA73-co-MMA27) than on P(DMAEMA78-co-MMA22) (Figure 3E). Our results suggest that surface charge can alter the composition and conformation of adsorbed proteins in the most superficial layer on the substrates.

4.2. Comparison of cell adhesion between short and long incubation periods


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In this study, 2 types of cell lines, HT-1080 and HeLa cells, were used for the experiments. HT1080 and HeLa cells are derived from mesenchymal cells (human fibrosarcoma), and epithelial cells (human cervix epithelioid carcinoma), respectively. Because the compositions of the extracellular matrix (ECM) are different between mesenchymal ECM (i.e., stromal ECM) and epithelial ECM [40], the cell adhesion receptors (i.e., integrin isoforms and non-integrin receptors) in HT-1080 and HeLa cells might be different. Thus, we used these 2 cell lines in this study. Particularly, it is expected that HT-1080 cells possess fibronectin and vitronectin receptors at higher levels than HeLa cells because fibronectin and vitronectin are stromal ECM against mesenchymal cells, such as HT-1080 cells, but not epithelial cells, such as HeLa cells. Thus, it is expected that HT-1080 can more easily adhere on serum proteins adsorbed substrates that might contain fibronectin and vitronectin. However, we expected that the tendency of hydrophobic and electrostatic interaction might not be different between HT-1080 and HeLa cells even though these interaction degrees can be changed. Many previous studies have assessed cell adhesion after long incubation periods (3 h or longer) [13-18]. In this study, we compared adherent cells after a long incubation (1 day) and short incubation (30 min or less). After the long incubation, few differences in adherent cell number were observed between P(MA73-co-MMA27) and P(DMAEMA78-co-MMA22) in serumcontaining medium. In contrast, there were evident differences between P(MA73-co-MMA27) and P(DMAEMA78-co-MMA22) following the short incubation (Figure 4 and Figure 5). These results indicate that the analysis of cell adhesion following a short incubation is required to elucidate cell adhesion mechanisms. On bare PSt, the number of adhered HeLa cells was smaller than that of HT-1080 cells in serumcontaining medium (Figure 4). On bare NT-PST in serum-containing medium, there are adsorbed


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stromal ECM proteins (fibronectin and vitronectin) as cell adhesion ligands (Figure 3). In mesenchymal HT-1080, it is expected that the cells possess the receptors against these proteins at higher level than in epithelial HeLa cells. Thus, the number of adhered HT-1080 cells was higher than that of adhere HeLa cells on bare PSt in serum-containing medium.

4.3. Cell adhesion in serum-free medium It has been generally believed that positively charged substrates can promote cell adhesion via the interaction with the negatively charged cell surface. If the negative charge of the cells strongly influenced cell adhesion, it is expected that negatively charged substrates suppress cell adhesion by electrostatic repulsion. However, both HT-1080 and HeLa cells adhered on negatively charged P(MA73-co-MMA27) as well as positively charged P(DMAEMA78-coMMA22) even in short-period incubations (Figure 4, Figure 5B and 5D). To elucidate the adhesion mechanism on P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt, we first focused on integrin dependency. However, the inhibitory cell adhesion assay with EDTA demonstrated that the cells adhered on these substrates without integrin in serum-free medium (Figure 6). For further elucidation, SCFS was performed (Figure 7). The adhesion force was lower on bare NT-PSt than P(MA73-co-MMA27) and P(DMAEMA78-co-MMA22), which exhibited larger absolute values of zeta potential than bare NT-PSt. Comparing P(MA73-coMMA27) and P(DMAEMA78-co-MMA22), P(MA73-co-MMA27) exhibited a higher adhesion force than P(DMAEMA78-co-MMA22). These results indicated that cell adhesion to electrically charged substrates cannot be simply accounted for by total electrical charge of the cell surface, although the electrical charge of the substrate surface can accelerate cell adhesion.


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Additionally, adherent cell numbers were similar in serum-free medium (Figure 5B and 5D), although the cell adhesion forces were different among the substrates (Figure 7). This might be explained by the set of the force threshold for maintaining cell adhesion. It is hypothesized that cells can keep adhering on the substrates during the cell adhesion assay when the adhesion force is over the threshold. In this study, we cannot identify the molecules that contribute to cell adhesion on P(MA73-coMMA27) and P(DMAEMA78-co-MMA22) in serum-free medium. However, it is expected that counter-ionic molecules and regions on the cell membrane will contribute to cell adhesion (e.g., sulfate groups in glycosaminoglycans for P(DMAEMA78-co-MMA22) and cationic amino acids for P(MA73-co-MMA27)). In addition to the electrostatic polymer substrates, cell adhesion was examined on nonionic polymer substrate (i.e., bare NT-PSt). Cells can adhere on bare NT-PSt (Figure 5B and 5D) but the adhesion force was lower than those on electrostatic polymer substrates (Figure 7). These results suggested that hydrophobic interaction was enough for cell adhesion but was weaker than electrostatic interaction. Thus, the cells mainly adhered on P(MA73-co-MMA27) and P(DMAEMA78-co-MMA22) in serum-free medium via electrostatic interaction rather than hydrophobic interaction.

4.4. Cell adhesion in serum-containing medium It is generally believed that cell adhesion is facilitated by serum proteins adsorbed on polymer substrates [19, 25, 27, 41]. However, the numbers of adherent cells on P(MA73-co-MMA27) and bare NT-PSt were lower in serum-containing medium than serum-free medium over a short


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incubation period (Figure 5), although proteins were adsorbed on their surfaces (Figure 3A-3D). SCFS revealed that the adhesion force was significantly reduced in serum-containing medium compared to serum-free medium, with the exception of the HT-1080 cell adhesion force on P(DMAEMA78-co-MMA22), which is speculated due to the difference between mesenchymal cells and epithelial cells (Figure 7). It has been suggested that adsorbed proteins act as an inhibitory factor for cell adhesion rather than a promotive factor. It is well known that albumin inhibits cell adhesion [26, 41]. Thus, the inhibitory effect of albumin on cell adhesion was strongly exerted on P(MA73-co-MMA27) and bare NT-PSt. In contrast to P(MA73-co-MMA27) and bare NT-PSt, cells began to adhere on P(DMAEMA78-coMMA22) within 5 min (Figure 5A and 5C), although the amount of adsorbed vitronectin and conformationally changed fibronectin was lower than on P(MA73-co-MMA27) and bare NT-PSt (Figure 3C and 3E). An inhibitory cell adhesion assay using EDTA revealed that cells can adhere on P(DMAEMA78-co-MMA22) even in the presence of EDTA, suggesting that the cells adhered via an integrin-independent mechanism (Figure 6). The integrin-independent adhesion mechanism seemed to facilitate rapid adhesion on P(DMAEMA78-co-MMA22). In this study, we could not identify the integrin-independent adhesion mechanisms. However, we propose several possible mechanisms. One is via the electrical force of P(DMAEMA78-co-MMA22), however the force is not accountable because both positive and negative charges can promote cell adhesion, as shown by the occurrence of cell adhesion in serum-free medium (Figures 5 and 7). Furthermore, integrin-independent adhesion mechanisms were not observed on P(MA73-coMMA27), which can also exhibit a negative electrical force (Figure 6). Another possible mechanism involves the specific interaction between adsorbed serum proteins and non-integrin cell adhesion receptors such as syndecans [29]. In addition, there are other mechanisms,


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including non-specific interactions, that we cannot exclude as possibilities. Characterizing the nature of integrin-independent adhesion mechanisms is the one of the biggest problems to be solved in the future.

4.5. Relationship of electrostatic interaction and protein adsorption with cell adhesion Charged polymers are widely used to control cell adhesion. Thus, cell adhesion profiles are often examined from the viewpoint of electrostatic potential and protein adsorption [37, 38, 41]. In contrast to previous studies, we characterized cell adhesion using cell biological methods (i.e., an EDTA inhibitory assay and SCFS) in addition to examining electrostatic potential and protein adsorption. A summary of our results is shown in Table 1. In serum-free medium, the cell adhesion forces on P(MA73-co-MMA27) and P(DMAEMA78-coMMA22) were greater than those of NT-PSt, which is accounted for by the existence of electrostatic interactions. Furthermore, the forces on P(MA73-co-MMA27) were greater than those on P(DMAEMA78-co-MMA22), even though negative charge can serve as a repulsive force due to the negative charge of the cell membrane. This result strongly suggests that the interaction between cells and charged substrates is not simply due to the negative charge of the cells. Protein adsorption has also been focused to explain cell adhesion on electrically charged substrates [37, 38, 41]. However, the amount of protein adsorption and conformationally changed fibronectin does not account for cell adhesion in serum medium. To examine cell adhesion over a short period of time, other factors must be considered. Studying non-integrinmediated adhesion on P(DMAEMA78-co-MMA22) will help to identify the other factors that influence cell adhesion. It might also be helpful to determine how albumin is adsorbed on the


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substrates [12, 41] because the cell adhesion force decreased on P(MA73-co-MMA27) and bare PSt.

5. Conclusion Cell adhesion on electrically charged substrates has been examined thoroughly from the viewpoints of electrostatic force and protein adsorption. In this study, we examined cell adhesion on these substrates using cell biological methods, as well as electrostatic interaction and protein adsorption. In serum-free medium, the cells strongly adhered on both negatively and positively charged substrates. These results indicate that the interaction between cells and charged substrates is not accounted for simply by the negative charge of the cells. In serum-containing medium, neither the electrostatic force nor protein adsorption is accountable for cell adhesion. Other factors (non-integrin cell adhesion receptors, etc.) must therefore be considered to understand cell adhesion in serum-containing medium.


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Figure 1. Experimental design used to distinguish various cell adhesion mechanisms.


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Figure 2. Chemical structures of the polymers (A) P(MA73-co-MMA27) and (B) P(DMAEMA78co-MMA22).


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Figure 3. Protein adsorption on the substrates in serum-containing medium. (A) The amount of total adsorbed protein measured by µBCA. (B-D) The amounts of adsorbed (B) albumin, (C) vitronectin, and (D) total fibronectin measured by ELISA with specific antibodies. (E) The amounts of conformationally-changed fibronectin measured by ELISA with an HFN7.1 antibody. MA, DMAEMA, and PSt indicate P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt, respectively. The data represent means ± SD (n=5). *, ***: P < 0.05, 0.005 vs. P(DMAEMA78-co-MMA22).


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Figure 4. Cell adhesion after 1 day in culture. (A) HT-1080 and (B) HeLa cells. MA, DMAEMA, and PSt indicate P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt, respectively. The data represent means ± SD (n=3).


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Figure 5. Time profile of cell adhesion. (A, B) HT-1080 cell adhesion in (A) serum-containing and (B) serum-free media. (C, D) HeLa cell adhesion in (C) serum-containing and (D) serumfree media. MA, DMAEMA, and PSt indicate P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NT-PSt, respectively. The data represent means ± SD (n=3).


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Figure 6. Inhibitory adhesion assay of (A) HT-1080 and (B) HeLa cells with EDTA. MA, DMAEMA, and PSt indicate P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NTPSt, respectively. The data represent means ± SD (n=3). *, **: P < 0.05, 0.01 vs. EDTA (-). N.S. indicates no significant difference between adhesion with and without EDTA.


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Figure 7. Adhesion force of (A) HT-1080 and (B) HeLa cells measured by SCFS. MA, DMAEMA, and PSt indicate P(MA73-co-MMA27), P(DMAEMA78-co-MMA22), and bare NTPSt, respectively. The data represent means ± SD (n=9-16). ***: P < 0.005 vs. FBS (-).


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Table 1. Summary of the experiments performed in this study. Polymer

FBS(+) Protein

FBS(-) Cell

adsorption adhesion

Cell

Adhesion

Cell

Cell

adhesion

mechanism

adhesion

adhesion

force P(MA73-co-

+++

+

MMA27) P(DMAEMA78- ++

+++

force

Very

Integrin-

weak

dependent

Strong

Both

++

Strong

++

Medium

++

Weak

integrin-

co-MMA22)

dependent and independent Bare NT-PSt

+++

-

Very

Integrin-

weak

dependent

Note: +++: Abundant; ++: Medium; +: Low; -: None


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ASSOCIATED CONTENT Cell shapes on charged polymer substrates after 30 min; Schematic illustration of protein adsorption on charged polymer substrates. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Takashi Hoshiba Frontier Center for Organic Materials, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510 Japan Tel: +81-238-26-3585, E-mail: [email protected]

Author Contributions C.Y. synthesized and characterized the polymers. K.S. characterized the polymer-coated substrates. T.H. evaluated protein adsorption and performed the cell experiments. All authors contributed to data analysis and to the writing of the manuscript.

Funding Sources This work was supported by a Grant-in-Aid for Young Scientists (A) (26702016) funded by MEXT, Japan.


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ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Young Scientists (A) (26702016) funded by MEXT, Japan.


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8. Hoshiba, T.; Nikaido, M.; Tanaka, M. Characterization of the Attachment Mechanisms of Tissue-Derived Cell Lines to Blood-Compatible Polymers. Adv. Healthcare Mater. 2014, 3(5) 775-784. 9. Hoshiba, T.;Nemoto, E.; Sato, K.; Orui, T.; Otaki, T.; Yoshihiro, A.; Tanaka, M. Regulation of the Contribution of Integrin to Cell Attachment on Poly(2-methoxyethyl Acrylate) (PMEA) Analogous Polymers for Attachment-Based Cell Enrichment. PLoS One 2015, 10(8) e0136066. 10. Cao, B.; Peng, Y.; Liu, X.; Ding, J. Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces. ACS Appl. Mater. Interfaces. 2017, 9(28) 23574-23585. 11. Iijima K.; Suzuki, R.; Iizuka, A.; Ueno-Yokohata, H.; Kiyokawa, N.; Hashizume, M. Surface Functionalization of Tissue Culture Polystyrene Plates with Hydroxyapatite Under Body Fluid Conditions and its effect on Differentiation Behaviors of Mesenchymal Stem Cells. Colloids Surf. B Biointerfaces 2016, 147, 351-359. 12. Hoshiba, T.; Yoshihiro A.; Tanaka, M. Evaluation of Initial Cell Adhesion on Poly(2methoxyethyl Acrylate) (PMEA) Analogous Polymers. J. Biomater. Sci. Polym. Edn. 2017, 28(10-12), 986-999. 13. Chang, H.-Y.; Kao, W.-L.; You, Y.-W.; Chu, Y.-H.; Chu, K.-J.; Chen, P.-J.; Wu, C.-Y.; Lee, Y.-H.; Shyue, J.-J. Effect of Surface Potential on Epithelial Cell Adhesion, Proliferation and Morphology. Colloids Surf. B Interfaces 2016, 141, 179.


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14. Chang, H.-Y.; Huang, C.-C.; Lin, K.-Y.; Kao, W.-L.; Liao, H.-Y.; You, Y.-W.; Lin, J.-H.; Kuo, Y.-T.; Kuo, D.-Y.; Shyue, J.-J. Effect of Surface Potential on NIH3T3 Cell Adhesion and Proliferation. J. Phys. Chem. C 2014, 118(26) 14464-14470. 15. Webb, K.; Hlady, V.; Tresco, P.A. Relative Importance of Surface Wettability and Charged Functional Groups on NIH3T3 Fibroblast Attachment, Spreading, and Cytoskeletal Organization. J. Biomed. Mater. Res. 1998, 41(3) 422-430. 16. Lee, J.H.; Jung, H.W.; Kang, I.-K.; Lee, H.B. Cell Behavior on Polymer Surfaces with Different Functional Groups. Biomaterials 1994, 15(9) 705-711. 17. Steele, J.G.; Dalton, B.A.; Johnson, G.; Underwood, P.A. Adsorption of Fibronectin and Vitronectin onto PrimariaTM and Tissue Culture Polystyrene and Relationship to the Mechanism of Initial Attachment of Human Vein Endothelial Cells and BHK-21 Fibroblasts. Biomaterials 1995, 16(14) 1057-1067. 18. McKeehan, W.L.; Ham, R.G. Stimulation of Clonal Growth of Normal Fibroblasts with Substrata Coated with Basic Polymers. J. Cell Biol. 1976, 71(3) 727-734. 19. Grinnell, F.; Feld, M.K. Fibronectin Adsorption on Hydrophilic and Hydrophobic Surfaces Detected by Antibody Binding and Analyzed During Cell Adhesion in Serum-Containing Medium. J. Biol. Chem. 1982, 257(9) 4888-4893. 20. Yoshikawa, C.; Hashimoto, Y.; Hattori, S.; Honda, T.; Zhang, K.; Terada, D.; Kishida, A.; Yoshinobu, T.; Kobayashi, H. Suppression of Cell Adhesion on Well-Defined Concentrated Polymer Brushes of Hydrophilic Polymers. Chem. Lett. 2010, 39(2) 142-143.


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21. Hirata, M.; Yamaoka, T. Effect of Stem Cell Niche Elasticity/ECM Protein on the SelfBeating Cardiomyocyte Differentiation of Induced Pluripotent Stem (iPS) Cells at Different Stages. Acta Biomater. 2018, 65(1) 44-52. 22. Gronowicz, G.; McCarthy, M.B. Response of Human Osteoblasts to Implant Materials: Integrin-Mediated Adhesion. J. Orthop. Res. 1996, 14(6) 878-887. 23. Wang, C.-C.; Lu, J.-N.; Young, T.-H. The Alteration of Cell Membrane Charge After Cultured on Polymer Membranes. Biomaterials 2007, 28(4) 625-631. 24. Mattinger, C.; Nyugen, T.; Schäfer, D.; Hörmann, K. Evaluation of Serum-Free Culture Conditions for Primary Human Nasal Epithelial Cells. Int. J. Hyg. Environ. Health 2002, 205(3) 235-238. 25. Dewez, J.L.; Doren, A.; Schneider, Y.J.; Rouxhet, P.G. Competitive Adsorption of Proteins: Key of the Relationship Between Substratum Surface Properties and Adhesion of Epithelial Cells. Biomaterials 1999, 20(6) 547-559. 26. Hoshiba, T.; Cho, C.S.; Murakawa, A.; Okahata, Y.; Akaike, T. The Effect of Natural Exracellular Matrix Deposited on Synthetic Polymers on Cultured Primary Hepatocytes. Biomaterials 2006, 27(26) 4519-4528. 27. García, A.J. Get s Grip: Integrins in Cell-Biomaterial Interactions. Biomaterials 2005, 26(36) 7525-7529. 28. Higuchi, A.; Kumar, S.S.; Ling, Q.-D.; Alarfaj, A.A.; Munusamy, M.A.; Murugan, K.; Hsu, S.-T.; Benelli, G.; Umezawa, A. Polymeric Design of Cell Culture Materials That Guide the


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Differentiation of Human Pluripotent Stem Cells. Prog. Polym. Sci. 2016, DOI: 10.1016/j.progpolymsci.2016.09.002. 29. Hozumi, K.; Otagiri, D.; Yamada, Y.; Sasaki, A.; Fujimori, C.; Wakai, Y.; Uchida, T.; Katagiri, F.; Kikkawa, Y., Nomizu, M. Cell Surface Receptor-specific Scaffold Requirements for Adhesion to Laminin-derived Peptide-chitosan Membranes. Biomaterials 2010, 31(12) 32373243. 30. Helenius, J.; Heisenberg, C.-P.; Gaub, H.E.; Muller, D.J. Single-Cell Force Spectroscopy. J. Cell Sci. 2008, 121(11) 1785-1791. 31. Saade, H.; León-Gómez, R.D.; Enríquez-Medrano, F.J.; López, R.G. Preparation of Ultrafine Poly(methyl methacrylate-co-methacrylic acid) Biodegradable Nanoparticles Loaded with Ibuprofen. J Biomater. Sci. Polym. Ed. 2016, 27(11) 1126-1138. 32. Zheng, J.Y.; Tan, M.J.; Thoniyot, P.; Loh, X.J. Unusual Thermogelling Behavior of Poly[2(dimethylamino)ethyl Methacrylate] (PDMAEMA)-Based Polymers Polymerized in Bulk. RSC Adv. 2015, 5 62314-62318. 33. Ishihara, K.; Oshida, H.; Endo, Y.; Watanabe, A.; Ueda, T.; Nakabayashi, N. Effects of Phospholipid Adsorption on Nonthrombogenicity of Polymer with Phospholipid Polar Group. J. Biomed. Mater. Res. 1993, 27(10) 1309-1314. 34. Okano. T.; Suzuki, K.; Yui, N.; Sakurai, Y.; Nakahama, S. Prevention of Changes in Platelet Cytoplasmic Free Calcuim Levels by Interaction with 2-Hydroxyethyl Methacrylate/Styrene Block Copolymer Surfaces. J Biomed. Mater. Res. 1993, 27(12), 1519-1525.


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35. ExPASy –Compute pI/Mw tool. https://web.expasy.org/compute_pi/ (accesed March 1, 2018). 36. Ugarova, T.P.; Zamarron, C.; Veklich, Y.; Bowditch, R.D.; Ginsberg, M.H.; Weisel, J.W,.; Plow, E.F. Conformational Transition in the Cell Binding Domain of Fibronectin. Biochemistry 1995, 34(13) 4457-4466. 37. Kesekowsky, B.G.; Collard, D.M.; García, A.J. Surface Chemistry Modulates Fibronectin Conformation and Directs Integrin Binding and Specificity to Control Cell Adhesion. J. Biomed. Mater. Res. 2003, 66A(2) 247-259. 38. Lin, J.-H.; Chang, H.-Y.; Kao, W.-L.; Lin, K.-Y.; Liao, H.-Y.; You, Y.-W.; Kuo, Y.-T.; Kuo, D.-Y.; Chu, K.-J.; Chu, Y.-H.; Shyue, J.-J. Effects of Surface Potential on Extracellular Matrix Protein Adsorption. Langmuir 2014, 30(34) 10328-10335. 39. Yan, Y.; Yang, H.; Su, Y.; Qiao, L. Albumin Adsorption on CoCrMo Alloy Surfaces. Sci. Rep. 2015, 5 18403. 40. Adachi, E.; Hopkinson, I.; Hayashi, T. Basement-Membranes Stromal Relationships: Interactions between Collagen Fibrils and the Lamina Densa. Int. Rev. Cytol. 1997, 173 73-156. 41. Arima, Y.; Iwata, H. Preferential Adsorption of Cell Adhesive Proteins From Complex Media on Self-Assembled Monolayers and its Effect on Subsequent Cell Adhesion. Acta Biomater. 2015, 26 72-81.


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SYNOPSIS (Word Style “SN_Synopsis_TOC”).


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Figure 1 487x400mm (72 x 72 DPI)


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Figure 2 143x149mm (72 x 72 DPI)


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Figure 3 245x279mm (300 x 300 DPI)


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Figure 4 274x100mm (300 x 300 DPI)


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Figure 5 199x120mm (300 x 300 DPI)


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Figure 6 163x214mm (300 x 300 DPI)


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Figure 7 116x189mm (300 x 300 DPI)


Characterization of Initial Cell Adhesion on ... - ACS Publications

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