Linker-free covalent attachment of the extracellular matrix protein tropoelastin to a polymer surface for directed cell spreading

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Acta Biomaterialia 5 (2009) 3371–3381 www.elsevier.com/locate/actabiomat

Linker-free covalent attachment of the extracellular matrix protein tropoelastin to a polymer surface for directed cell spreading Daniel V. Bax a,b,*, David R. McKenzie a, Anthony S. Weiss b, Marcela M.M. Bilek a a b

Applied and Plasma Physics, School of Physics, University of Sydney, Building A28, Sydney, NSW 2006, Australia School of Molecular and Microbial Biosciences, University of Sydney, Building G08, Sydney, NSW 2006, Australia Received 4 February 2009; received in revised form 2 April 2009; accepted 12 May 2009 Available online 20 May 2009

Abstract Polymers are used for the fabrication of many prosthetic implants. It is desirable for these polymers to promote biological function by promoting the adhesion, differentiation and viability of cells. Here we have used plasma immersion ion implantation (PIII) treatment of polystyrene to modify the polymer surface, and so modulate the binding of the extracellular matrix protein tropoelastin. PIII treated, but not untreated polystyrene, bound tropoelastin in a sodium dodecyl sulfate (SDS)-resistant manner, consistent with previous enzymebinding data that demonstrated the capability of these surfaces to covalently attach proteins without employing chemical linking molecules. Furthermore sulfo-NHS acetate (SNA) blocking of tropoelastin lysine side chains eliminated the SDS-resistant binding of tropoelastin to PIII-treated polystyrene. This implies tropoelastin is covalently attached to the PIII-treated surface via its lysine side chains. Cell spreading was only observed on tropoelastin coated, PIII-treated polystyrene surfaces, indicating that tropoelastin was more biologically active on the PIII-treated surface compared to the untreated surface. A contact mask was used to pattern the PIII treatment. Following tropoelastin attachment, cells spread preferentially on the PIII-treated sections of the polystyrene surface. This demonstrates that PIII treatment of polystyrene improves the polymer’s tropoelastin binding properties, with advantages for tissue engineering and prosthetic design. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Plasma immersion ion implantation; Tropoelastin; Covalent linkage; Polystyrene; Cell spreading

1. Introduction Polymers are used for the fabrication of many prosthetic implants such as vascular grafts, heart valves, bone implants and joints, parenchyma and in reconstructive surgery [1,2]. Some biopolymers used for tissue engineering are required to be inert in order to prevent biological activity, e.g. for joint prosthesis, intraocular lenses and bloodcontacting devices, whereas it is desirable for others, such as for bone and skin implants, to promote biological function by promoting the adhesion, differentiation and viability of cells [2]. Extracellular matrix (ECM) proteins * Corresponding author. Address: Applied and Plasma Physics, School of Physics, University of Sydney, Building A28, Sydney, NSW 2006, Australia. Tel.: +61 2 9351 7333; fax: +61 2 9351 5858. E-mail address: [email protected] (D.V. Bax).

mediate cell attachment signals that can be utilized in the generation of novel hybrid biomaterials [3]. Polymer surfaces are typically either too hydrophobic or negatively charged to adhere cells directly, and therefore many polymers are modified to enhance cell binding. Physical methods of surface modification are appealing for manufacture, and include electrostatic treatment [4], carbon deposition [5], UV/gamma irradiation [6,7], plasma discharge [8] and ion implantation [1,9–11]. These surfaces bind to cells via receptor and non-receptor mechanisms, and so may not elicit the cell signals that cells would receive from ECM proteins [2]. Cellular interactions with ECM are vital for cell survival and tissue maintenance and are involved in many biological functions such as cell migration and proliferation, tissue organisation, wound repair, development and host immune responses [12]. A lack of these signals often results in cellular dedifferentiation and

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.05.016

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apoptosis. The ability to covalently attach biologically active ECM proteins to the surface enables biological control of cellular activity [13]. Simple physisorption of ECM proteins, as often used to coat surfaces with molecules such as fibronectin and collagen, is non-specific, relying on multiple weak hydrophobic, van der Waals or permanent dipole interactions. Binding through these interactions results in variable extents of attachment, persistence and conformational stability [14,13]. Covalent interactions offer the opportunity to present the attached ECM proteins in a conformationally more relevant state. Furthermore, strongly fixing proteins to surfaces enhances the resorption resistance of the protein. Many methods employed for covalent protein linkage require chemical linker molecules such as disulfides, silanization, epoxides or glutaraldehyde [15–17], which can result in chemical modification of active residues, may have adverse biological effects and involve more complex surface attachment processes. In order to overcome problems associated with the use of whole proteins, short synthetic peptides are often used to improve cell adhesion. Examples include the integrinbinding peptide RGD [18,19] and the proteoglycan-based peptide KRSR [20], but these peptides do not have the full functionality and receptor specificity of the native ECM protein [14]. For example, many ECM proteins contain synergy sites [21] which are required for integrin specificity, and/or multiple receptor binding sites which are required for cellular signalling [22]. However a major barrier to the use of ECM molecules is material-induced random folding which may sterically hinder binding [23]. Unlike previous studies which explored the direct cell adhesive activity of plasma ion immersion implantation (PIII)-treated surfaces, in this paper, we explore the use of PIII to provide a patternable platform for enhanced cell spreading through the attachment of an ECM protein, tropoelastin. We have chosen the PIII methodology as it has been previously shown to allow linker-free covalent attachment of bioactive molecules to polymer surfaces [24,25]. Polystyrene was chosen because it is the most common material for culturing cells, and is the most widely applied surface for studying cell–material interactions. We have chosen the ECM protein tropoelastin to direct cell interactions because it interacts with a range of biologically relevant cell surface receptors [26–28]. 2. Materials and methods 2.1. Materials Biaxially oriented 0.1 mm thick polystyrene sheets were obtained from Goodfellows. Recombinant tropoelastin was produced in-house as described in Ref. [28]. The mouse anti-human elastin antibody, BA-4, and the goat antimouse IgG-HRP conjugated secondary antibodies were purchased from Sigma. Human dermal fibroblasts (HDFs) were sourced from the Coriell Research Institute (Camden,

NJ, USA). Unless stated otherwise all other reagents were purchased from Sigma. 2.2. PIII treatment Polystyrene sheets were cut into 0.8 cm  8 cm strips and wiped with 100% ethanol. Samples were mounted onto a substrate holder, covered by an electrically connected mesh and immersed in an inductively coupled radiofrequency (RF) plasma. Pulses of high voltage bias were applied to the substrate holder as in PIII. The RF power was 100 W. The working gas pressure was 2 mtorr of high-purity nitrogen with a flow rate of 72 standard cubic centimeters (sccm). The samples were PIII treated by applying 20 kV pulses lasting for 20 ls with a repetition rate of 50 Hz to the sample holder for 800 s. The sample holder was earthed between the pulses. Further details of the treatment process and its application to the surface attachment of bioactive enzymes can be found in Refs. [24,30,31]. Untreated controls did not undergo treatment in the plasma chamber. A contact mask in the form of 3 mm wide ADH Kapton blocking adhesive tape (Associated Gaskets, Australia) was used to limit PIII treatment to specific regions of the polystyrene. After PIII treatment the tape was removed. 2.3. SNA blocking Tropoelastin was blocked with sulfo-NHS acetate (SNA) as previously described [29]. Briefly, tropoelastin was solubilized in 100 mM NaHCO3, pH 8.5 to 1 mg ml1 and a 25-fold molar excess of SNA (Pierce) was added and incubated at room temperature for 1 h. After incubation the excess SNA was removed by dialysis against four 1 l volumes of phosphate-buffered saline (PBS) at 4 °C. A control without SNA was included alongside the SNA-treated sample. Following dialysis the absorbance at 280 nm was measured and used to determine the tropoelastin concentration. 2.4. ELISA Strips of untreated and PIII-treated polystyrene were cut into 0.8 cm  0.8 cm squares and placed into the wells of a 24-well plate (Greiner). SNA-treated or untreated tropoelastin was diluted to the appropriate concentration in PBS and 0.75 ml added per well and incubated at 4 °C for 16 h. Unbound tropoelastin was removed by aspiration, and the samples were washed with 3  1 ml aliquots of PBS. The samples were washed by transferring to 1.5 ml 0.05% Tween (v/v) in PBS for 10 min at room temperature, 1.5 ml of 1 M NaOH for 10 min at room temperature, or to 1.5 ml of 5% SDS (w/v) in PBS and incubated at 90 °C for 10 min. Non-treated samples were washed in 3  1 ml PBS. The samples were returned to the 24-well plate and washed with 3  1 ml PBS. Non-specific binding to the polystyrene was blocked with 3% (w/v) bovine serum albumin (BSA) in

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PBS for 1 h at room temperature. Following BSA blocking the samples were washed with 2  1 ml PBS, then incubated in 0.75 ml of 1:2000 diluted mouse anti-elastin antibody (BA-4) for 1 h at room temperature. The antibody was removed, and the samples were washed in 3  1 ml PBS before incubation in 0.75 ml of 1:10,000 diluted goat anti-mouse IgG-HRP conjugated secondary antibody for 1 h at room temperature. The secondary antibody was removed and the samples washed with 4  1 ml PBS. The samples were transferred to a new 24-well plate and 0.75 ml ABTS solution (40 mM ABTS (2,20 -azino-bis(3ethylbenzthiazoline-6-sulfonic acid)), 0.01% (v/v) H2O2 in 0.1 M NaOAc, 0.05 M NaH2PO4, pH 5) was added. After 30–40 min the plates were agitated and 100 ll aliquots of the ABTS were transferred to a 96-well plate and the absorbance was read at 405 nm using a plate reader.

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and the cell pellets were resuspended in 5 ml warm serum-free media. The cell densities were counted and adjusted to 1  105 cells ml1. The BSA blocking solution was aspirated from the wells, followed by 3  1 ml washes PBS. 0.75 ml aliquots of cells were added to the wells, and placed at 37 °C in a 5% CO2 incubator for 90 min. For masking experiments the cells were placed at 37 °C in a 5% CO2 incubator for 60 min, washed with 2  1 ml serum-free media, then incubated for a further 60 min at 37 °C in a 5% CO2 incubator. The cells were immediately fixed with the addition of 81 ll of 37% (w/v) formaldehyde directly to the well for 20 min. The formaldehyde was aspirated, and the wells filled with PBS before layering a glass plate onto the 24-well plate. The level of cell spreading was determined by phase-contrast microscopy. Cells were defined as spread when phase dark with visible nuclei, and non-spread when rounded and phase bright.

2.5. Spectrometry 3. Results Untreated and PIII-treated polystyrene were cut into 6 cm  0.8 cm strips and placed into 15 ml Falcon tubes. SNA-treated or untreated tropoelastin was diluted to the appropriate concentration in PBS and 8 ml added per tube and incubated at 4 °C for 16 h. Unbound tropoelastin was removed by aspiration, and the samples were washed with 3  10 ml volumes of PBS. Samples were SDS treated by transferring to 8 ml of 5% SDS (w/v) in PBS and incubated at 90 °C for 10 min. Following SDS treatment, the samples were placed into a new 15 ml tube and washed with 3  10 ml PBS. Non-SDS-treated samples were washed with 3  10 ml PBS. Samples were dried using dry air flow prior to accumulation of spectra using a Digilab FTS7000 Fourier transform infrared (FTIR) spectrometer fitted with an attentuated total reflection (ATR) accessory with a trapezium germanium crystal and an incidence angle of 45°. To obtain sufficient signal/noise ratio and resolution of spectral bands, 500 scans with a resolution of 1 cm1 were taken. Difference spectra, used to detect changes associated with the presence of tropoelastin, were obtained by subtracting spectra of treated samples not incubated in tropoelastin from the spectra of those incubated in tropoelastin solution. All spectral analysis was performed using GRAMS software. 2.6. Cell spreading analysis 0.8 cm  0.8 cm squares of untreated and PIII-treated polystyrene were incubated in 0.75 ml tropoelastin diluted to 20 lg ml1 in PBS in a 24-well plate at 4 °C for 16 h. Unbound tropoelastin was aspirated and the samples were washed with 3  1 ml PBS. Non-specific polystyrene binding was blocked with 10 mg ml1 heat denatured BSA (80 °C for 10 min, then cooled on ice) in PBS for 1 h at room temperature. Near-confluent 75 cm2 flasks of human skin fibroblasts were trypsinized by incubating with trypsin–EDTA at 37 °C for 4 min, followed by neutralization with an equal volume of 10% serum-containing media. The cell suspensions were centrifuged at 800g for 3 min,

3.1. Enhanced tropoelastin binding to PIII-treated polystyrene Fig. 1A shows the level of tropoelastin that bound to untreated and PIII-treated polystyrene after incubation in 0–50 lg ml1 tropoelastin solutions as measured by ELISA. Maximal binding occurred at a concentration of 5 lg ml1 for both untreated and PIII-treated polystyrene. No increase in tropoelastin attachment to the polystyrene surface was observed at concentrations above 5 lg ml1. At all concentrations, the amount of tropoelastin that bound to PIII-treated polystyrene was higher than on the untreated polystyrene. Absorbances of 0.27 and 0.2 above background levels were observed for PIII-treated and untreated polystyrene, respectively, at a tropoelastin coating concentration of 5 lg ml1. To confirm that maximum surface binding of tropoelastin occurred at a coating concentration of 5 lg ml1, ATR FTIR analysis was used to examine PIII-treated and untreated polystyrene that had been immersed in a range of tropoelastin concentrations (Fig. 1B). Subtraction of the spectrum from a control sample not exposed to tropoelastin, yielded peaks at wavenumbers associated with amide I and amide II bands (1545 cm1, 1654 cm1) [32]. These peaks are associated with vibrations of the amide bonds in tropoelastin, and therefore with the presence of tropoelastin on the surface. In agreement with the ELISA results, PIII-treated and untreated polystyrene each showed an increase in the amplitude of the amide I and II peaks with increasing tropoelastin concentration up to 5 lg ml1 in the incubation solution. No further increase in amplitude was observed up to a concentration of 50 lg ml1 in agreement with the ELISA data. As no additional tropoelastin bound to either the PIIItreated or untreated polystyrene surfaces at concentrations above 5 lg ml1, a concentration of 10 lg ml1 in the incubation solution was used for all subsequent assays unless stated otherwise.

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A

B

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Fig. 1. (A) ELISA detection of tropoelastin bound to untreated (grey, diamonds) and PIII-treated (black, squares) polystyrene using a range of tropoelastin coating concentrations. No-tropoelastin absorbances of 0.108 and 0.126 were deducted for untreated and PIII-treated polystyrene, respectively. Error bars indicate standard deviations of triplicate measurements. (B) ATR FTIR spectrophotometric detection of tropoelastin bound to untreated (left) and PIII-treated (right) polystyrene using a range of tropoelastin coating concentrations (indicated on the right of the graphs). The traces were scaled, and then a no-tropoelastin trace was deducted from the sample traces. The traces were separated to allow easy visualization by the addition of 0.005 to the 0.5 lg ml1 trace, 0.01 to the 1 lg ml1 trace, 0.015 to the 2 lg ml1 trace, 0.02 to the 5 lg ml1 trace, 0.025 to the 10 lg ml1 trace, and 0.03 to the 50 lg ml1 trace. The amide peaks are indicated by arrows.

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3.2. PIII treatment prevents the SDS-mediated dissociation of tropoelastin from polystyrene

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of 0.08 and 0.16, respectively. This indicates that the mechanism of tropoelastin binding is different on untreated and PIII-treated polystyrene.

In order to explore the mechanism of interaction of tropoelastin with the untreated and PIII-treated polystyrene surfaces, samples were washed with dissociating solutions and the amount of tropoelastin present was measured. Uncoated and tropoelastin-coated samples were subjected to 0.05% Tween washing, 5% SDS washing at 90 °C, washing in 1 M NaOH, or PBS washing. As the BA-4 anti-tropoelastin antibody used is not sensitive to the conformational state of tropoelastin [33], the amount of tropoelastin present was measured by ELISA (Fig. 2). In the absence of tropoelastin, untreated and PIII-treated polymer gave background absorbances in the range 0.078–0.085 for all of the above dissociating washing methods. Tropoelastin bound to untreated polystyrene with an absorbance of 0.12, and to PIII-treated polystyrene with an absorbance of 0.155 when washed with PBS. Within error bars of a no-treatment control, tropoelastin was not dissociated from untreated or PIII polystyrene with 0.05% Tween or 0.1 M NaOH washes, giving absorbances of 0.12 and 0.135, respectively, for untreated polystyrene and 0.148 and 0.155 for PIII-treated polystyrene. In contrast, washing at 90 °C in 5% SDS dissociated tropoelastin from untreated polystyrene but did not dissociate tropoelastin from PIII-treated polystyrene, giving absorbances

3.3. SNA blocks SDS-resistant binding of tropoelastin to treated polystyrene Amines, and in particular lysine, residues on tropoelastin were preblocked with SNA prior to incubation with the polystyrene samples. The samples were SDS washed at 90 °C and the amount of tropoelastin remaining was detected by ELISA. Controls that had not been exposed to tropoelastin gave background absorbances of 0.11 and 0.14 for untreated and PIII-treated polystyrene, respectively (Fig. 3A). Consistent with Fig. 2, washing for 10 min at 90 °C in 5% SDS completely removed tropoelastin from untreated polystyrene to background levels, but did not remove tropoelastin from PIII-treated samples, giving absorbances of 0.01 and 0.13 above background levels, respectively. In contrast, SNA-blocked tropoelastin was completely removed from both untreated and PIII-treated polystyrene by SDS washing, as indicated by absorbances of 0.05 and 0.05 above background levels, respectively. This result is consistent with the interpretation that SNA blocks the sites on the tropoelastin molecule that are involved in the SDS-resis-

0.18 PIII treated – tropoelastin PIII treated + tropoelastin 0.16

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Fig. 2. Resistance of tropoelastin binding to untreated (grey) and PIII-treated polystyrene (black) to washing conditions as measured by ELISA assay. No-tropoelastin controls are shown by dashed bars, and 10 lg ml1 tropoelastin-coated samples are shown by solid bars. Washing conditions are shown under each bar. Error bars indicate standard deviations of triplicate measurements.

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A

As SNA blocks amine groups, this suggests that amines on lysine side chains participate in the SDS-resistant binding of tropoelastin to PIII-treated polystyrene. 3.4. PIII treatment enhances cell spreading on tropoelastin

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Wavenumber (nm) Fig. 3. (A) ELISA detection of SDS-resistant binding of SNA treated (grey bar), or untreated tropoelastin (black bar) to untreated (left) and PIII-treated (right) polystyrene. No-tropoelastin control absorbances (0.11 for untreated polystyrene, 0.13 for PIII-treated polystyrene) were deducted. Error bars indicate standard deviations of triplicates. (B) ATR FTIR spectrometric detection of SDS-resistant binding of SNA-treated (grey line), or untreated tropoelastin (black line) bound to PIII-treated polystyrene. The traces were scaled, and then a no-tropoelastin trace was deducted from the sample traces. The traces were separated to allow easy visualization by the addition of 0.008 to the no-SNA block trace. The amide peaks are indicated by arrows.

tant interaction of tropoelastin with PIII-treated polystyrene. To ensure that the lack of SDS-resistant binding observed was not due to SNA blocking interfering with the ELISA antibody epitope, ATR FTIR spectrometry was used to detect the SDS resistance of SNA-treated and untreated tropoelastin binding to PIII-treated polystyrene (Fig. 3B). In the absence of SNA, the amide I and II peaks were observed at 1654 and 1545 cm1; however, in the presence of SNA there were no amide peaks, confirming that SNA blocked the SDS-resistant binding of tropoelastin to PIII-treated polystyrene.

PIII treatment affects the mechanism of protein binding to the polymer surface. Therefore the cell spreading activity of tropoelastin covalently bound to PIII-treated polystyrene and tropoelastin non-covalently bound to untreated polystyrene was measured as described in Ref. [34] (Fig. 4A). Cells were allowed to spread onto the tropoelastin-coated surfaces for 90 min to reduce the possibility of cell spreading onto cell-synthesized ECM proteins, rather than the tropoelastin coating. Human skin fibroblasts did not spread on BSA blocked, untreated polystyrene in the absence of tropoelastin. Tropoelastin coating only marginally increased cell spreading to 4% after incubation in 20 lg ml1 tropoelastin solution. BSA-blocked PIII-treated polystyrene supported 8% cell spreading. Tropoelastin coating dramatically increased cell spreading on the PIIItreated surfaces up to 83% after incubation in 20 lg ml1 tropoelastin solution. Phase-contrast microscopy showed that human skin fibroblasts spreading onto BSA blocked, untreated or PIII-treated polystyrene in the absence of tropoelastin coating were rounded and phase bright (Fig. 4B). However, the fibroblasts were flattened and phase dark when spreading on 20 lg ml1 tropoelastin-coated PIII polystyrene. In contrast, the fibroblasts remained rounded and phase bright when spreading onto tropoelastincoated untreated polystyrene. Therefore the ability of bound tropoelastin to support cell spreading is dramatically enhanced when it is bound to PIII-treated polystyrene. 3.5. Patterning of fibroblast spreading by PIII masking and tropoelastin coating Masking during PIII treatment of polystyrene was used to direct PIII treatment, and as a consequence cell spreading to specific areas of the polystyrene surface. Following tropoelastin coating and BSA blocking of the patterned surfaces the degree of human skin fibroblast spreading was observed by phase-contrast microscopy (Fig. 5). Fibroblasts were phase dark and spread on the PIII-treated regions of the polystyrene. In contrast, fibroblasts on the masked (untreated) polystyrene regions were rounded and phase bright. Furthermore, if the samples were washed gently during cell spreading, the cells were removed from the masked (untreated) surface regions, but remained on the PIII-treated regions. Therefore a masked PIII treatment followed by incubation with tropoelastin and subsequent blocking with BSA was found to be an effective method of directing cell spreading to predefined areas of the polystyrene surface.

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Fig. 4. (A) Percentage of human dermal fibroblast spreading onto untreated (grey bars) or PIII-treated (black bars) samples. Cell spreading was observed by phase-contrast microscopy with cells being counted as spread when flattened with phase dark areas and unspread when round and phase bright. The samples were incubated in 0 lg ml1 (left), or 20 lg ml1 (right) tropoelastin solutions and subsequently blocked with BSA. No bar is observed in the untreated 0 lg ml1 tropoelastin sample as there was 0% cell spreading. Error bars indicate standard deviations of triplicates. (B) Phase-contrast photographs of human dermal fibroblasts spreading onto PIII-treated (top) or untreated (bottom) samples which were incubated in 0 lg ml1 (left), or 20 lg ml1 (right) tropoelastin solution and subsequently BSA blocked.

4. Discussion In order for biomaterials to integrate into a biological system they should ideally promote biological function by supporting the adhesion, differentiation and viability of cells [2]. As the ECM protein tropoelastin supports cell adhesion and signalling [26–28] we have investigated the binding of tropoelastin to PIII-treated polystyrene and its effect on cell spreading. Using ELISA detection of tropoelastin, we observed an increased level of tropoelastin binding to PIII-treated

polystyrene compared to untreated polystyrene at all tropoelastin concentrations. Binding saturated at a concentration of 5 lg ml1 tropoelastin for both untreated and PIIItreated polystyrene. This result was confirmed by ATR FTIR spectrometry which showed saturation of the amide I and II peaks at 5 lg ml1, indicating that the binding capacity of the surface also saturates at this concentration. This indicates that the saturation observed in the ELISA assay is due to the amount of tropoelastin present, rather than saturation of antibodies or the detection reagent used.

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Fig. 5. Phase-contrast photographs of human skin fibroblasts spreading onto masked PIII-treated polystyrene surfaces coated with tropoelastin by incubation in 10 lg ml1 tropoelastin solution. Above the dashed line is a masked (untreated) area and below the dashed line the sample was PIII treated. The left sample was washed with media after 60 min of the 120 min spreading incubation, whereas the right sample was not washed during the 120 min spreading incubation.

The increased level of tropoelastin binding to PIII-treated polymer as compared to the untreated control indicates a higher density of tropoelastin on the surface. Previous work assessing protein attachment to PIII-treated polystyrene showed [35] that horseradish peroxidase (HRP) formed a homogeneous layer of protein with 100% surface coverage as compared to a patchy coverage of about 30% on the untreated surface. The increased tropoelastin bound to the PIII-treated surface is therefore likely to indicate a more complete coverage of the surface. Adsorption of ECM proteins can be affected by physical and chemical properties such as wettability, electrical charge, surface roughness, topography, pH, or the presence of chemical groups such as carbon, amine or oxygen groups [2,36]. PIII has been shown to break bonds in polymers, resulting in the loss of hydrogen and the formation of high-energy free radicals and conjugated regions in the polymer [37]. The radicals can react with the polymer chains, resulting in a sub-surface amorphous carbon layer, and with atmospheric oxygen forming oxidized structures such as esters, carbonyl, carboxyl and hydroxyl groups [38–40]. This results in increased polarity and wettability which would be expected to modify physisorption processes for proteins. Washing with a variety of dissociating solutions prior to tropoelastin detection was used to investigate attachment mechanisms. Tropoelastin was removed from the untreated surface by incubation at 90 °C in 5% SDS solution for 10 min. In contrast, the PIII-treated surface retained tropoelastin under these conditions. The BA-4 anti-tropoelastin antibody used in the ELISA assay is not sensitive to tropoelastin conformation [33] as validated by the detection of tropoelastin on PIII-treated polystyrene after SDS washing. SDS resistance of tropoelastin-binding to PIII-treated polystyrene is consistent with previous data showing SDS resistance of HRP and catalase binding to PIII-treated surfaces [24,25]. SDS is a detergent that is used to unfold proteins [41]. Since SDS interferes with the physical forces which result in physisorption of proteins onto surfaces

but does not attack covalent bonds, leaving the protein’s primary structure intact, SDS washing has been used as a method to test whether proteins are covalently attached to surfaces [42–45] and to detect covalently bound drug– protein adducts [46]. In some situations, steric hindrance may prevent the SDS from accessing all of the sites where physical forces bind the protein and surface. An example of such a situation may be where there is a thick coverage of strongly denatured and aggregated protein completely blocking access to the interface with the surface. Since our control is the more hydrophobic of the surfaces tested and SDS successfully removed all of the protein from it, it is unlikely that steric hindrance could be responsible for the SDS-resistant binding observed on PIII-treated surfaces. Therefore we propose that tropoelastin binds to PIII-treated polystyrene via covalent linkages. Blocking of candidates was used as a way to test which groups on the protein participate in the SDS-resistant binding. In previous work, Tris pre-incubation reduced the SDS-resistant binding of soy bean peroxidase to PIII-treated surfaces [24], indicating that C–NH2 or C–OH groups on the protein participate in the covalent binding. Since SNA blocks amine groups and not OH groups, our SNA blocking data show that amine groups are involved in tropoelastin binding to PIII-treated surfaces. Although there may be other groups capable of similar interactions, including the amino-terminus, because tropoelastin has such a high concentration of lysine side chains this mechanism would be expected to dominate as observed. Cell-spreading assays were used to test the bioactivity of the bound tropoelastin. Tropoelastin can bind directly to cells through several cell surface receptors, including the elastin binding protein, cell surface glycosaminoglycans and integrin avb3 [26–28]. Binding of cells to tropoelastin results in cell morphological changes [47,48], chemotaxis [49], decreased cell proliferation [50] and angiogenesis [51]. We have observed differences in the binding mechanism of tropoelastin to PIII-treated and untreated polystyrene surfaces and this may affect its ability to interact with

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cells. Therefore we investigated the cell-spreading properties of tropoelastin-coated PIII-treated and untreated polystyrene surfaces. We BSA blocked all surfaces after incubation in tropoelastin to prevent direct cell interaction with the underlying surfaces. Additionally we conducted the spreading assays for 90 min to reduce cell-derived ECM synthesis, thereby ensuring that cells spread only to the bound tropoelastin. We found a dramatic increase in cell spreading on tropoelastin-coated surfaces when the surfaces were PIII treated. The observed increase in cell spreading may be due to either a different conformation of tropoelastin when bound to a PIII-treated surface rather than an untreated surface, or alternatively may simply be a result of the increased loading of tropoelastin induced by the treatment. Tropoelastin may adhere in a cell-adhesive conformation on PIII-treated compared to untreated polystyrene. Atomic force microscopy and circular dichroism studies have suggested that the forces generated by protein–substrate binding may be sufficient to disrupt the protein structure [52,53]. Furthermore extremely hydrophobic surfaces can bind large quantities of protein; however, these proteins can become rigid, and so constrain their accessibility for cell binding [54,13]. Pristine polystyrene is hydrophobic, but PIII treatment results in a hydrophilic, more polar surface. This could reduce the rigidity of tropoelastin, and so increase its cell-adhesive properties. Previous work showed increased longevity of catalytic activity in surface-attached enzymes such as catalase [25], HRP [30] and SBP [24] on PIII-treated polyethylene as compared to untreated surfaces. This indicates that the PIII-treated surfaces provide a local environment that better retains proteins in their active conformation than the hydrophobic untreated surface. Our tropoelastin cell-spreading results are consistent with the affect of surface properties on integrin engagement onto fibronectin [55,56]. Integrin engagement is strongly dependent upon the conformation of fibronectin [13,57]. Integrin engagement onto fibronectin is known to follow the following trend [56]: a highly hydrophilic, polar uncharged surface shows greater engagement than a hydrophilic negatively or positively charged surface, which in turn shows greater engagement than a hydrophobic surface. As seen for fibronectin, cell binding to tropoelastin is via integrins and cell surface GAGs [27,28]. This suggests that surface properties would be expected to mediate the interaction of tropoelastin with cells and therefore its ability to support cell spreading. As PIII treatment makes the surface more hydrophilic and polar, it is likely that it will assist in integrin-mediated cell spreading to tropoelastin. A simple contact mask was used during PIII treatment to direct this treatment to specific areas of the polymer surface. Following tropoelastin coating and BSA blocking, we were able to direct cell spreading to specific regions of the polystyrene surface. PIII patterning is a straightforward technique for directed cell adhesion and may have many applications for spatially influencing cell

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behaviour on biomaterials. For example, it is possible to encourage cell adhesion and tissue integration into areas of an implant requiring integration, while simultaneously inhibiting cell adhesion to other areas in which cell adhesion would be detrimental. Refinement of patterns on a planar surface to include smaller and more complex features for use in biosensors or lab-on-chip devices would be straightforward, relying on techniques commonly employed in the semiconductor industry. Since the PIII method is non-line-of-sight, it can easily be applied to treat three-dimensional objects with complex surfaces, such as orthopaedic implants and tissue engineering scaffolds. 5. Conclusions This paper examined tropoelastin binding to a polystyrene surface, treated by a previously reported [30,31,24,25] plasma treatment process, which uses energetic ion bombardment as applied through PIII. The motivation was to influence cell spreading through the action of the surface-adhered ECM protein tropoelastin. The PIII-treated surface were found to attach significantly more protein than the untreated controls upon incubation in solutions containing 0–50 lg ml1 tropoelastin. The surface coverage saturated at a concentration of 5 lg ml1 tropoelastin. The binding mechanism is believed to be covalent because the protein was not removed by SDS at 90C, and to involve lysine side-chain groups because it can be blocked by incubating the tropoelastin in SNA prior to its exposure to the surface. Tropoelastin attached to the PIII-treated surfaces supported dramatically increased cell spreading, around 80%, as compared with the same cells in contact with tropoelastin coated onto an untreated control polystyrene surface. Since the surfaces were BSA blocked after tropoelastin incubation and BSA-blocked surfaces of both kinds without attached tropoelastin showed spreading levels of less than 8%, we conclude that the cellular responses are associated with the increased amount or improved cell-adhesive conformation of the tropoelastin on the PIII-treated surface rather than with the condition of the underlying surface. This result is consistent with our previous observations of activity of enzymes attached to polymer surfaces subject to the same treatment process [24,25,30,31]. In all of these works the attached enzymes, which were also resistant to removal by SDS, showed enhanced enzyme activity compared to the untreated controls. This is indicative of improved protein stability on the PIII-treated polymer surfaces. The potential of our surface treatment process to produce patterned surfaces for directing cell spreading was demonstrated by applying it through a contact mask. Lines of demarcation with respect to cell spreading corresponded exactly with the edges of the mask. Since the process is readily applied to three-dimensional scaffolds we conclude that it may be suitable for use in tissue engineering or for

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surface

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Acknowledgements The authors acknowledge support from the Australian Research Council. References [1] Bacakova L, Walachova K, Svorcik V, Hnatowicz V. Adhesion and proliferation of rat vascular smooth muscle cells (VSMC) on polyethylene implanted with O+ and C+ ions. J Biomater Sci Polym Ed 2001;12:817–34. [2] Bacakova L, Filova E, Rypacek F, Svorcik V, Stary V. Cell adhesion on artificial materials for tissue engineering. Physiol Res 2004;53(Suppl. 1):S35–45. [3] Stephan S, Ball SG, Williamson M, Bax DV, Lomas A, Shuttleworth AC, et al. Cell-matrix biology in vascular tissue engineering. J Anat 2006;204:495–502. [4] Bowlin GL, Rittgers SE, Milsted A, Schmidt SP. In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts. J Vasc Surg 1998;27:504–11. [5] Kaibara M, Iwata H, Wada H, Kawamoto Y, Iwaki M, Suzuki Y. Promotion and control of selective adhesion and proliferation of endothelial cells on polymer surface by carbon deposition. J Biomed Mater Res 1996;31:429–35. [6] Heitz J, Svorcik V, Bacakova L, Rockova K, Ratajova E, Gumpenberger T, et al. Cell adhesion on polytetrafluoroethylene modified by UV-irradiation in an ammonia atmosphere. J Biomed Mater Res A 2003;67:130–7. [7] Gumpenberger T, Heitz J, Bauerle D, Kahr H, Graz I, Romanin C, et al. Adhesion and proliferation of human endothelial cells on photochemically modified polytetrafluoroethylene. Biomaterials 2003;24:5139–44. [8] Alves CM, Yang Y, Carnes DL, Ong JL, Sylvia VL, Dean DD, et al. Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption. Biomaterials 2007;28:307–15. [9] Svorcik V, Rybka V, Hnatowicz V, Bacakova L, Lisa V, Kocourek F. Surface properties and biocompatibility of ion-implanted polymers. J Mater Chem 1995;5:27–30. [10] Lee JS, Kaibara M, Iwaki M, Sasabe H, Suzuki Y, Kusakabe M. Selective adhesion and proliferation of cells on ion-implanted polymer domains. Biomaterials 1993;14:958–60. [11] Manso M, Navas CR, Gilliland D, Ruiz PG, Rossi F. Cellular response to oxygen containing biomedical polymers modified by Ar and He implantation. Acta Biomater 2007;7:735–43. [12] Danen EHJ, Sonnenberg A. Integrins in regulation of tissue development and function. J Pathol 2003;201:632–41. [13] Garcia AJ, Vega MD, Boettiger D. Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation. Mol Biol Cell 1999;10:785–98. [14] Garcia AJ. Get a grip: integrins in cell–biomaterial interactions. Biomaterials 2005;26:7525–9. [15] Cutler SM, Garcia AJ. Engineering cell adhesive surfaces that direct integrin alpha(5)beta(1) binding using a recombinant fragment of fibronectin. Biomaterials 2003;24:1759–70. [16] Huang LL, Lee PC, Chen LW. Comparison of epoxides on grafting collagen to polyurethane and their effects on cellular growth. J Biomed Mater Res 1998;39:630–6. [17] Khor E. Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials 1997;18:95–105. [18] Larsen CC, Kligman F, Koltke-Marchant K, Marchant RE. The effect of RGD fluorosurfactant polymer modification of ePTFE on

[20]

[21]

[22] [23] [24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34] [35]

[36]

[37] [38]

[39]

endothelial cell adhesion, growth, and function. Biomaterials 2006;27:4846–55. Lin YS, Wang SS, Chung TW, Wang YH, Chiou SH, Hsu JS, et al. Growth of endothelial cells on different concentrations of Gly-ArgGly-Asp photochemically grafted in polyethylene glycol modified polyurethane. Artif Organs 2001;25:617–21. Dee KC, Andersen TT, Bizios R. Design and function of novel osteoblast-adhesive peptides for chemical modification of biomaterials. J Biomed Mater Res 1998;40:371–7. Obara M, Kang MS, Yamada KM. Site-directed mutagenesis of the cell-binding domain of human fibronectin—separable, synergistic sites mediate adhesive function. Cell 1988;53:649–57. Rosso F, Giordano A, Barbarisi M, Barbarisi A. From cell–ECM interactions to tissue engineering. J Cell Physiol 2004;199:174–80. Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering. Biomaterials 2003;24:4353–64. MacDonald C, Morrow R, Weiss AS, Bilek MMM. Covalent attachment of functional protein to polymer surfaces: a novel onestep dry process. J R Soc Interface 2008;5:663–9. Nosworthy NJ, Ho JPY, Kondyurin A, McKenzie DR, Bilek MMM. The attachment of catalase and poly-L-lysine to plasma immersion ion implantation-treated polyethylene. Acta Biomater 2007;3:695–704. Mecham RP, Hinek A, Entwistle R, Wrenn DS, Griffin GL, Senior RM. Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein. Biochemistry 1989;264:16652–7. Broekelmann TJ, Kozel BJ, Ishibashi H, Werneck CC, Keeley FW, Zhang L, et al. Tropoelastin interacts with cell-surface glycosaminoglycans via its COOH-terminal domain. J Biol Chem 2005;280:40939–47. Rodgers UR, Weiss AS. Integrin alpha(v)beta(3) binds a unique nonRGD site near the C-terminus of human tropoelastin. Biochimie 2004;86:173–8. Wu WJ, Vrhovski B, Weiss AS. Glycosaminoglycans mediate the coacervation of human tropoelastin through dominant charge interactions involving lysine side chains. J Cell Biol 1999;274:21719–24. Ho JPY, Nosworthy NJ, Bilek MMM, Gan BK, McKenzie DR, Chu PK, et al. Plasma-treated polyethylene surfaces for improved binding of active protein. Plasma Process Polym 2007;4:583–90. Gan BK, Nosworthy NJ, McKenzie DR, dos Remedios CG, Bilek MMM. Plasma immersion ion implantation treatment of polyethylene for enhanced binding of active horseradish peroxidase. J Biomed Mater Res A 2007;85:605–10. Green RJ, Hopkinson I, Jones RAL. Unfolding and intermolecular association in globular proteins adsorbed at interfaces. Langmuir 1999;5:5102–10. Grosso LE, Scott M. Peptide sequences selected by BA4, a tropoelastin-specific monoclonal-antibody, are ligands for the 67-kilodalton bovine elastin. Biochemistry 1993;32:13369–74. Humphries MJ. Cell-substrate adhesion assays. Curr Protoc Cell Biol 1998:9.1.1–9.1.11. Gan BK, Kondyurin A, Bilek MMM. Comparison of protein surface attachment on untreated and plasma immersion ion implantation treated polystyrene: protein islands and carpet. Langmuir 2007;23:2741–6. Wang YX, Robertson JL, Spillman WB, Claus RO. Effects of the chemical structure and the surface properties of polymeric biomaterials on their biocompatibility. Pharm Res 2004;2:1362–73. Chipara MI. ESR investigations on ion beam irradiated polymers. Nucl Instrum Methods Phys Res B 1997;131:85–90. Kondyurin A, Gan BK, Bilek MMM, Mizuno K, McKenzie DR. Etching and structural changes of polystyrene films during plasma immersion ion implantation from argon plasma. Nucl Instrum Methods Phys Res B 2006;251:413–8. Kondyurin A, Gan BK, Bilek MMM, McKenzie DR, Mizuno K, Wnher R. Argon plasma immersion ion implantation of polystyrene films. Nucl Instrum Methods Phys Res B 2008;266:1074–84.

D.V. Bax et al. / Acta Biomaterialia 5 (2009) 3371–3381 [40] Gan BK, Bilek MMM, Kondyurin A, Mizuno K, McKenzie DR. Etching and structural changes in nitrogen plasma immersion ion implanted polystyrene films. Nucl Instrum Methods Phys Res B 2006;247:254–60. [41] Laemmli UK. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 1970;227:680. [42] Vandenberg E, Elwing H, Askendal A, Lundstrom I. Protein immobilization to 3-aminopropyl triethoxy silane glutaraldehyde surfaces—characterization by detergent washing. J Colloid Interface Sci 1991;143:327–35. [43] Shlyakhtenko LS, Gall AA, Weimer JJ, Hawn DD, Lyubchenko YL. Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate. Biophys J 1999;77:568–76. [44] Hodneland CD, Lee YS, Min DH, Mrksich M. Selective immobilization of proteins to self-assembled monolayers presenting active sitedirected capture ligands. PNAS 2002;99:5048–52. [45] Bohnert JL, Fowler BC, Horbett TA, Hoffman AS. Plasma gas discharge deposited fluorocarbon polymers exhibit reduced elutability of adsorbed albumin and fibrinogen. J Biomater Sci Polym Ed 1990;1:279–97. [46] Zhou S. Separation and detection methods for covalent drug–protein adducts. J Chromatogr B 2003;797:63–90. [47] Karnik SK, Wythe JD, Sorensen L, Brooke BS, Urness LD, Li DY. Elastin induces myofibrillogenesis via a specific domain, VGVAPG. Matrix Biol 2003;22:409–25. [48] Karnik SK, Brooke BS, Bayes-Genis A, Sorensen L, Wythe JD, Schwartz RS, et al. A critical role for elastin signaling in vascular morphogenesis and disease. Development 2003;130: 411–23.

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[49] Senior RM, Griffin GL, Mecham RP. Chemotactic responses of fibroblasts to tropoelastin and elastin-derived peptides. J Clin Invest 1982;70:614–8. [50] Jung S, Rutka JT, Hinek A. Tropoelastin and elastin degradation products promote proliferation of human astrocytoma cell lines. J Neutropathol Exp Neurol 1998;57:439–48. [51] Robinet A, Fahem A, Cauchard J-H, Huet E, Vincent L, Lorimier S, et al. Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP. J Cell Sci 2005;118:343–56. [52] Meadows PY, Bemis JE, Walker GC. Single-molecule force spectroscopy of isolated and aggregated fibronectin proteins on negatively charged surfaces in aqueous liquids. Langmuir 2003;19:9566–72. [53] Norde W, Favier JP. Structure of adsorbed and desorbed proteins. Colloid Surf 1992;64:87–93. [54] Groth T, Altankov G, Kostadinova A, Krasteva N, Albrecht W, Paul D. Altered vitronectin receptor (alpha(v) integrin) function in fibroblasts adhering on hydrophobic glass. J Biomed Mater Res 1999;44:341–51. [55] Keselowsky BG, Collard DM, Garcia AJ. Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation. PNAS 2005;102:5953–7. [56] Keselowsky BG, Collard DM, Garcia AJ. Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion. J Biomed Mater Res A 2003;66:247–59. [57] Krammer A, Craig D, Thomas WE, Schulten K, Vogel VA. A structural model for force regulated integrin binding to fibronectin’s RGD-synergy site. Matrix Biol 2002;21:139–47.