Nano-Structured Cell-Adhesive and Cell-Repulsive Plasma-Deposited Coatings: Chemical and Topographical Effects on Keratinocyte Adhesion

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<ul><li><p>Nano-Structured Cell-AdCell-Repulsive Plasma-DeChemical and TopographKeratinocyte Adhesion</p><p>R,</p><p>Introduction expression.[35] In the last decades such knowledge is steering</p><p>l</p><p>composition and morphology. Such substrates could</p><p>possibly drive cell adhesion, growth and physiology in</p><p>,</p><p>,</p><p>,</p><p>-</p><p>,</p><p>Full Paper</p><p>E. Sardella, R. Gristina, G. S. Senesi</p><p>ave been plasma-deposited on poly(ethyleneterephthalate) surfaces previously structured with nano-metric conical features by meansof colloidal lithography. Surface analysis revealed that both coatings are conformal on nano-structured substrates, with their wettabilitydepending on the substrate morphology. Theeffect of surface chemistry and surface topogra-phy on cell adhesion has been investigated andclarified. The adhesion of a human keratinocytecell-line was found to be strongly dependent onthe surface topography for plasma-depositedacrylic acid (cell-adhesive), and on the surfacechemistry for poly(ethylene oxide)-like (cell-repul-sive) coatings.</p><p>540many applications, including biomaterials, prostheses</p><p>tissue/cell engineering, regenerative medicine, biosensors</p><p>microfluidics and biochips. So far, the development of</p><p>nano-structured biointerfaces was limited by the high</p><p>production cost of nano-features with advanced precise</p><p>methods (e.g. electron beam lithography[9]), by the</p><p>difficulty to pattern large area substrates, and by the long</p><p>patterning time. Newer approaches to nano-scale patterns</p><p>such as X-ray lithography[10] and nano-imprint lithogra</p><p>phy,[11,12] are thus being developed for obtaining reliable</p><p>Institute of Inorganic Methodologies and Plasmas (IMIP) CNR,70126 Bari, ItalyFax: 39 80 544 3405; E-mail: sardella@chimica.uniba.itL. Detomaso, R. dAgostino, P. FaviaDepartment of Chemistry, University of Bari, 70126 Bari, ItalyR. dAgostino, P. FaviaPlasma Solution Srl, Spin-off of the University of Bari,via Orabona 4, 70126 Bari, ItalyH. Agheli, D. S. SutherlandiNANO Interdisciplinary Research Center, University of Aarhus,Aarhus 8000, DenmarkCells respond to micro- and nano-metric surface fea-</p><p>tures[1,2] with changes in adhesion, morphology, and gene</p><p>a trend in biomedical research, aiming to affordable</p><p>techniques for producing micro- and nano-patterned</p><p>biomedical surfaces and scaffolds with controlled chemica[68]Cell-adhesive and cell-repulsive coatings hEloisa Sardella,* Loredana Detomaso,Hossein Agheli, Duncan S. SutherlandPlasma Process. Polym. 2008, 5, 540551</p><p> 2008 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheimhesive andposited Coatings:ical Effects on</p><p>oberto Gristina, Giorgio S. Senesi,Riccardo dAgostino, Pietro FaviaDOI: 10.1002/ppap.200800005</p></li><li><p>morphology of human keratinocytes. This investigation</p><p>modifications. More details on the CL step are in ref.[39] PET-CL</p><p>Nano-Structured Cell-Adhesive and Cell-Repulsive . . .fast and cheap fabrication techniques of nano-structured</p><p>large area surfaces, as required in cell culture and tissue</p><p>engineering applications. Colloidal lithography (CL),</p><p>among other techniques, revealed as inexpensive, well</p><p>established nano-fabrication technique able to generate</p><p>nano-scale features of different shapes on large areas at</p><p>reasonable speed and cost.[13,14] CL-structured interfaces</p><p>have been often used to produce substrates featured</p><p>ad hoc to investigate cell adhesion and morphology.[14,16]</p><p>CL exploits the self-assembly of micro/nano-metric</p><p>colloidal particles in hexagonal arrays on properly</p><p>prepared surfaces; such arrays act as physical masks,</p><p>and can be transferred at the surface of the substrate with</p><p>lithographic steps, including sputtering and etching</p><p>processes. Plasma-enhanced chemical vapor deposition</p><p>(PE-CVD)[15] can also be used to modify the substrate</p><p>surface through the openings of the sphere array.</p><p>Investigating the effects of nano-topography (rough-</p><p>ness, shape/size of features, geometric vs. random</p><p>distribution, etc.) on cell behavior requires cell-growth</p><p>experiments on flat and nano-structured surfaces char-</p><p>acterized by same identical chemical composition and</p><p>different topography, or vice versa.[16] Studies performed</p><p>on surfaces with different topography and identical</p><p>chemical properties show that also topography can control</p><p>the organization of adsorbed proteins directly involved in</p><p>cell adhesion processes.[17,18]</p><p>How to use micro/nano-topography, independently</p><p>from surface chemistry, to drive the adhesion and growth</p><p>of cells, and to which extent cell functions (e.g., production</p><p>of certain proteins) could be stimulated/inhibited by</p><p>selected chemical/topographic surface features are still</p><p>open issues in this field.</p><p>Plasma processes are investigated and utilized in a</p><p>growing number of applications in life science, from</p><p>biomaterials to tissue engineering, from sterilization to</p><p>biosensors.[19] Plasma processes can be coupled with CL</p><p>techniques[2024] to promote the self-assembly of the beads</p><p>when the substrate is not able to properly support particle</p><p>crystallization, to transfer the pattern of the assembled</p><p>layer to the substrate by means of PE-CVD or plasma</p><p>etching processes and to modify, in a conformal and</p><p>homogeneous way, the chemistry of the patterned surface.</p><p>PE-CVD of thin organic stable films, in particular, can</p><p>functionalize surfaces in a specific and controlled way,</p><p>with the aim of tuning adhesion, spreading and prolifera-</p><p>tion of cells on surfaces from cell-adhesive to cell-</p><p>repulsive.[2527] Surfaces with different oxygen-containing</p><p>functionalities affect adhesion and spreading of different</p><p>cell lines;[2831] in particular, plasma-deposited acrylic acid</p><p>(pdAA) films with controlled surface density of carboxyl</p><p>groups induce attachment and growth of keratinocytes,</p><p>osteoblasts and fibroblasts,[3234] while plasma-depositedpoly(ethylene oxide)-like (PEO-like) coatings with at least</p><p>Plasma Process. Polym. 2008, 5, 540551</p><p> 2008 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheimsamples were blown with N2 to remove any particulate</p><p>contamination, and cleaned in 70% ethanol-water. Fabrication</p><p>and cleaning were carried out in a class-1000 clean room before</p><p>packaging in airtight boxes for transfer.</p><p>Plasma Deposition Processes</p><p>PdAA coatings were deposited in a stainless steel plasma reactor</p><p>with two internal stainless steel parallel plate circular ( 25 cm)</p><p>electrodes; the upper is shielded and connected to a RF (13.56 MHz)</p><p>generator through a manual matching network, the lower isaims to highlight the role of surface chemistry and of</p><p>surface topography in cell adhesion, but also to stress how</p><p>surface chemical/morphological properties of materials</p><p>can be adjusted to drive the behavior of cells.</p><p>Experimental Part</p><p>Colloidal Lithography</p><p>Poly(ethylene terephthalate), PET, has been utilized. PET-flat and</p><p>CL nano-structured (PET-CL) substrates have been plasma coated</p><p>with pdAA (PET-flat-pdAA; PET-CL-pdAA) and PEO-like (PET-flat-</p><p>PEO; PET-CL-PEO) coatings. The CL technique, described in detail</p><p>elsewhere,[10,37,38,39] utilizes electrostatically assembled, dispersed</p><p>monolayers of colloidal spherical particles as masks to transfer a</p><p>pattern at the surface of a substrate. PET (Goodfellow, UK) substrates</p><p>were pretreated with an O2 radio frequency (RF, 13.56 MHz) glow</p><p>discharge to improve the electrostatic self-assembly of a multilayer</p><p>of polyelectrolytes, i.e., poly(diallyldimethylammonium chloride)</p><p>(PDDA, MW 20 00035 0000 g mol1, Aldrich, UK), poly(sodium 4-styrenesulfonate) (PSS, MW 70 000 g mol1, Aldrich, UK), andaluminum chloride hydroxide (ACH, Reheis). Subsequent assem-</p><p>bly of a colloidal mask of sulfate-modified polystyrene (PS) beads</p><p>(1075 nm diameter, IDC, USA) from water solution, followed bydrying, resulted in a dispersed colloidal monolayer with short-</p><p>range order. The pattern of the mask was transferred at the PET</p><p>surface with a combination of vertical and angled Ar ionbombardment. A DC argon ion beam (CAIBE Ion Beam System,</p><p>Oxford Ionfab) was used to perform a two-step etching process as</p><p>follows: the first step used Ar ions (250 eV, 0.074 mA cm2,incident angle 158 off normal angle, sample rotation, 560 s) and3 sccm O2 released at the surface as a chemical etching assistance;</p><p>the second step employed Ar ions (250 V, 0.074 mA cm2,normal incident angle, 240 s) and 3 sccm O2.</p><p>The sputter-etching process was performed until the PS beads</p><p>were completely removed, resulting in conical pillars and chemical7080% of ether CH2CH2O over all carbon moieties</p><p>discourage protein and cell adhesion.[35,36]</p><p>For this work pdAA and PEO-like coatings have been</p><p>plasma-deposited on flat and conical CL-nano-structured</p><p>PET substrates, in order to study simultaneously surface</p><p>topographical and chemical effects on adhesion andgrounded. The reactor was evacuated with a rotary pump</p><p> 541</p></li><li><p>mass flow controllers. For both PE-CVD processes samples were</p><p>positioned on the ground electrode of the reactors.</p><p>during the measurements. WCA values reported are the mean of at</p><p>Cell culture experiments were performed with NCTC 2544 human</p><p>E. Sardella et al.</p><p>542Chemical and Morphological Analyses</p><p>Processed surfaces were characterized with X-ray photoelectron</p><p>spectroscopy (XPS) and water contact angle (WCA) measurements.</p><p>XPS analyses were performed with a PHI 5300 ESCA instrument</p><p>with non-monochromatized Mg Ka X-rays. Wide scan [01000 eV</p><p>binding energy (BE)] and high-resolution (C1s, O1s) spectra were</p><p>acquired at 458 electron take-off angle within 1 h after thedeposition. Error bars in the graphs are the standard deviations on</p><p>3 replicated samples. C1s spectra of pdAA and PEO-like coatings</p><p>were best fitted with four peak components corresponding to C-</p><p>atoms with zero, one, two and three carbon-oxygen bonds,</p><p>namely: C0 (CH, CC; 285.00.2 eV, BE reference); C1 (COH,COC; 286.6 0.2 eV); C2 (OCO, CO; 288.10.2 eV), C3(COOH, COOR; 289.10.2 eV). For PET-flat and PET-CL surfaces thefollowing C1s peak components were also used: Ca (aromatic CH,</p><p>CC, 284.70.2eV); C0a (CH in the terephthalate moiety,286.20.2 eV); C3a (COOR, 288.6 0.2 eV).[40] The best fittingprocedure was performed with a fixed full width at half maximum</p><p>(FWHM) of 2.00 eV and a 80100% Gaussian for all peak</p><p>components. Sample charging was corrected by positioning the</p><p>hydrocarbon C1s peak component at 284.7 eV (Ca) for PET-flat and</p><p>at 285.0 eV (C0) for the other substrates.</p><p>Static and dynamic WCA measurements were performed soon</p><p>after each deposition at room temperature, with double distilled</p><p>water, using a manual optical goniometer (Rame-Hart mod100-</p><p>00). Advancing WCA (ua) measurement were performed byequipped with a liquid N2 trap. A mixture of acrylic acid (AA,</p><p>Sigma Aldrich, 99%) vapors and argon (Air Liquide) was used as</p><p>feed (AA 3 sccm, Ar 5 sccm). The pressure, monitored with a MKS</p><p>baratron, was kept constant at 150 mTorr. Discharges were ignited</p><p>for 5 min at a power input of 100 W, resulting in pdAA films</p><p>30 5 nm thick on flat silicon samples. PdAA coatings deposited insuch conditions are known to induce good adhesion and growth</p><p>of fibroblasts[24] and were used to coat PET-flat and PET-CL</p><p>substrates.</p><p>PEO-like coatings were deposited in a stainless steel plasma</p><p>reactor equipped with two vertical parallel plate asymmetric</p><p>stainless steel electrodes The small ( 8 cm) one is connected to a</p><p>RF (1356 MHz) generator trough a matching network, the large (</p><p>18 cm) electrode is grounded. The reactor was evacuated with a</p><p>rotary/root pump system. A mixture of diethylene glycol dimethyl</p><p>ether (DEGDME, Sigma Aldrich) vapors and Ar was used as feed</p><p>(0.4 sccm DEGDME, 5 sccm Ar). The pressure, monitored with a</p><p>MKS baratron, was kept constant at 400 mTorr. Discharges were</p><p>ignited for 30 min at a power input of 5 W, resulting in PEO-like</p><p>films 305 nm thick on flat silicon samples. Only this set ofexperimental conditions was used, in this work, to coat PET-flat</p><p>and PET-CL substrates; PEO-like coatings deposited in such</p><p>conditions show net protein and cell repulsive non fouling</p><p>properties[25,33,34] on flat surfaces.</p><p>AA and DEGDME were degassed with freeze/thaw cycles and</p><p>used without further purification; vapors of both liquids were fed</p><p>from glass reservoirs with a needle valve, Ar was fed through MKSprogressively increasing the volume of the water drop by stepwise</p><p>Plasma Process. Polym. 2008, 5, 540551</p><p> 2008 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheimkeratinocyte cell lines from stocks routinely grown in Dulbeccos</p><p>modified eagle medium (DMEM) supplemented with 10% fetal</p><p>bovine serum, 50 IU mL1 penicillin, 50 IU mL1 streptomycinand 200 103 M glutamine, under 5% CO2/95% air atmosphere at37 8C. All samples were placed, modified side up, in 24 well cultureplates (Iwaki 24 wells). Cells were obtained after trypsinization of</p><p>confluent or near-confluent culture, seeded (1104 cells per well)in suspension on all test materials and incubated at 37 8C under5%CO2/95% air atmosphere. After different periods of incubation</p><p>(30 min, 180 min and 24 h) cells were fixed with formaldehyde</p><p>(4 wt.-% in PBS for 15 min, then stained with Comassie blue. Cell</p><p>attachment, distribution and morphology on flat and nano-</p><p>structured surfaces were measured as a function of culture time</p><p>using digital images acquired with a phase contrast light micro-</p><p>scope (Leica DM IL). The number of adhered cells was determined</p><p>in at least 10 areas of 0.8 mm2 per sample. At least three repeated</p><p>samples per experiment were analyzed. The two-way ANOVA and</p><p>the Bonferroni post test were used to evaluate statistical</p><p>significant differences among samples. Variations were consid-</p><p>ered significant when p</p></li><li><p>Nano-Structured Cell-Adhesive and Cell-Repulsive . . .</p><p>(a) nd of PET-CL (b) surfaces.shows the nc-mode AFM 3D topography images of PET-flat</p><p>Figure 1. 3D AFM micrographs and height distribution of PET-flat(a) and PET-CL (b) substrates with their normalized height</p><p>distribution. Only one height distribution was recorded on</p><p>PET-flat surfaces, with AH value of 4 2 nm. The conicalshape of PET-CL structures is well evident; two height</p><p>distributions were observed on PET-CL, the first peak</p><p>(lower value) refers to the AH of the background, the</p><p>second (higher) value refers to the AH of the conical nano-</p><p>features. The distance between the two peaks is the AH</p><p>value of the nano-cones, 117 5 nm in this work. C1s XPSsignals shown in Figure 2 evidence the different surface</p><p>composition of PET-flat and PET-CL surfaces, due to the</p><p>chemical changes (broken/re-arranged bonds) induced by</p><p>the Ar/O2 etching process in the CL procedure. The typical</p><p>shake-up feature (292 eV) due to the aromatic rings in PET</p><p>disappears from the outer layer of PET-CL surfaces, the</p><p>COOR/H (C3) component becomes less intense, and new</p><p>components C1 and C2 (ether/alcohol and carbonyl</p><p>groups) appear. A slight decrease of the O/C XPS ratio is</p><p>also observed, from 0.40 0.01 (PET-flat) to 0.37 0.2 (PET-CL). These results attest for a high crosslinking of the</p><p>outermost layers (up to 10 nm) of PET due to the Ar/O2sputter/etching process.[37,41]</p><p>Data in Table 1 show no significant difference of static</p><p>WCA values between PET-flat and PET-CL surfaces; a</p><p>marke...</p></li></ul>


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