Research Article www.acsami.org
Abrogated Cell Contact Guidance on Amino-Functionalized Microgrooves Caroline Mörke,† Henrike Rebl,† Birgit Finke,‡ Manuela Dubs,§ Peter Nestler,∥ Aissam Airoudj,⊥ Vincent Roucoules,⊥ Matthias Schnabelrauch,§ Andreas Körtge,# Karine Anselme,⊥ Christiane A. Helm,∥ and J. Barbara Nebe*,† †
Department of Cell Biology, University Medical Center Rostock, Schillingallee 69, 18057 Rostock, Germany Leibniz-Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Strasse 2, 17489 Greifswald, Germany § Biomaterials Department, INNOVENT e. V., Pruessingstrasse 27B, 07745 Jena, Germany ∥ Institute of Physics, University of Greifswald, Felix-Hausdorff-Strasse 6, 17487 Greifswald, Germany ⊥ Institute of Materials Sciences of Mulhouse (IS2M), CNRS UMR7361, 15 rue jean starcky, BP2488, 68057 Mulhouse cedex, France # Institute of Electronic Appliances and Circuits, University of Rostock, Albert-Einstein-Strasse 2, 18059 Rostock, Germany ‡
ABSTRACT: Topographical and chemical features of biomaterial surfaces affect the cell physiology at the interface and are promising tools for the improvement of implants. The dominance of the surface topography on cell behavior is often accentuated. Striated surfaces induce an alignment of cells and their intracellular adhesion-mediated components. Recently, it could be demonstrated that a chemical modification via plasma polymerized allylamine was not only able to boost osteoblast cell adhesion and spreading but also override the cell alignment on stochastically machined titanium. In order to discern what kind of chemical surface modifications let the cell forget the underlying surface structure, we used an approach on geometric microgrooves produced by deep reactive ion etching (DRIE). In this study, we systematically investigated the surface modification by (i) methyl-, carboxyl-, and amino functionalization created via plasma polymerization processes, (ii) coating with the extracellular matrix protein collagen-I or immobilization of the integrin adhesion peptide sequence Arg-Gly-Asp (RGD), and (iii) treatment with an atmospheric pressure plasma jet operating with argon/oxygen gas (Ar/O2). Interestingly, only the amino functionalization, which presented positive charges at the surface, was able to chemically disguise the microgrooves and therefore to interrupt the microtopography induced contact guidance of the osteoblastic cells MG-63. However, the RGD peptide coating revealed enhanced cell spreading as well, with fine, actin-containing protrusions. The Ar/O2-functionalization demonstrated the best topography handling, e.g. cells closely attached even to features such as the sidewalls of the groove steps. In the end, the amino functionalization is unique in abrogating the cell contact guidance. KEYWORDS: geometric microgrooves, RGD, physical plasma, water contact angle, zeta potential, contact guidance, actin cytoskeleton, correlative microscopy
1. INTRODUCTION
altered by modification of the implant surface and consequently properties such as surface energy, surface charge, and wettability. The creation of a biologically active implant surface involves the application of an additional layer onto the titanium surface using physiochemical and biochemical deposition techniques.4 A convenient method to attach functional organic groups onto surfaces is the deposition of a nanometer-thin plasma polymer. These groups can be e.g. methyl, carboxyl, and amino groups. Another way to chemically modify implant
Surface features such as topography or chemistry play an essential role in osteoblast adhesion and spreading on biomaterials. These processes determine the first phase of cell-biomaterial interaction. In consequence, the quality of this initial contact will influence the direct cell growth and implant covering, which is relevant for enhancement of bone ingrowth and, ergo, implant success. Advanced biomaterials should promote these specific cell responses.1 The cellular mechanisms affected by the surface chemistry and topography are not fully understood, but it is reported that the initial cell adhesion is more influenced by the surface chemistry than topography.2,3 For bone-replacing biomaterials, the osseointegration can be © 2017 American Chemical Society
Received: December 21, 2016 Accepted: March 6, 2017 Published: March 15, 2017 10461
DOI: 10.1021/acsami.6b16430 ACS Appl. Mater. Interfaces 2017, 9, 10461−10471
Research Article
ACS Applied Materials & Interfaces surfaces involves coating them with peptides or proteins.5 The tripeptide sequence Arg-Gly-Asp (RGD) has been identified in many cell adhesion proteins, such as fibronectin, collagen, and vitronectin. Cell adhesion on biomaterials is mediated by binding of the cells’ adhesion receptors, the integrins, to extracellular matrix (ECM) proteins containing the RGD peptide sequence. Consequently, synthetic RGD peptides have been applied or covalently bound to surfaces to improve cell adhesion.6 Collagen is the main ECM protein of bone tissue; it also facilitates cell adhesion via integrin binding and is considered the “gold standard” for cell-material investigations.7 However, it has been observed that hydrophobic surfaces exhibit a higher protein adsorption,8 although cells prefer moderate hydrophilic surfaces exhibiting a water contact angle of 40−60°.2,7−9 This is because the ECM proteins can be absorbed in a denatured state on hydrophobic surfaces making RGD-containing integrin-binding sites less accessible for the cells. Several other studies confirm that the chemical modification of an implant surface plays an important role in bone tissue engineering.10,11 Carboxyl groups lead to negatively charged surfaces and are reported to be a platform for the immobilization of cell adhesion molecules, proteins, and cellstimulating molecules, e.g. cytokines or fatty acids, which can stimulate cell behavior.12 Surface functionalization with amides improves the initial spreading step.13 Amide and carboxyl groups facilitate osteogenic differentiation, whereas in vivo experiments revealed that carboxyl-coated implants caused negative effects on the inflammatory reaction whereas the amino-functionalized implants did not.12 Besides surface chemistry modifications, a great deal of research activity has been addressed to surface topography modifications of titanium-based implants. Stochastic microrough surfaces were reported to promote a better mechanical bony fixation than smoother ones, whereas the initial cell number as well as spreading was reduced;1,2,8 but the stochastic structures possess also an inconsistency in the dimension and repetition of their surface topography variables, such as elevations, cavities, or ridges, which impede the identification of topography-induced cell responses.8 Therefore, geometrically designed, grooved structures with constant repetitive surface topography variables have been used. These microgrooved structures cause a strong topographically induced cell elongation, i.e. they promote contact guidance along the direction of groove.14,15 In this study, we chose to use geometrically microgrooves to investigate the impact of different chemistries on cells in contact with the biomaterial topography. The different surface functionalizations were performed by (i) methyl, carboxyl, and amino functionalization created via plasma polymerization processes, (ii) coating with the extracellular matrix protein collagen-I or immobilization of the integrin adhesion peptide sequence Arg-Gly-Asp (RGD), and (iii) treatment with an atmospheric pressure plasma jet operating with argon/oxygen gas (Ar/O2). Our objective was to determine whether the chemistry has a greater impact on the initial cell attachment, spreading, and elongation than the topography itself by reversing the topography-induced contact guidance. Preliminary studies showed that the amino functionalization of machined titanium predominantly affected cell orientation, spreading, and adhesion.7 Therefore, we wanted to determine whether this special plasma deposition with amino groups is unique in influencing the cell guidance, or if also other plasma depositions with their specific chemistries as well as the
immobilization of ECM proteins and peptide sequences cause similar cellular responses. Finally, this work led to a better knowledge of how chemical surface properties influence cell responses and can be used to design new biofunctional implant surfaces.
2. EXPERIMENTAL SECTION 2.1. Titanium Arrays. Periodically microtextured samples with regular groove geometry having a groove and plateau width of 20 μm and step height of 2 μm were used in these experiments. For sample fabrication, silicon wafers with a diameter of 10 × 10 mm and a thickness of 500 μm were microstructured using deep reactive-ion etching (DRIE) (Center for Microtechnologies ZFM, Germany).14 Unstructured silicon wafers were used as controls. The samples were finally sputter-coated with 100 nm titanium (Ti). 2.2. Chemical Surface Modifications. Collagen-I: The immobilization of collagen-I (Col) (Sigma-Aldrich, Germany) was performed with a wet chemical process by coating with collagen-I (rat, 20 mg/ cm2, BD Biosciences, USA) diluted to 200 mg/mL in 0.1% acetic acid (Sigma-Aldrich). The Col solution (0.1 mg/mm2) was put on the samples for 3 h, removed, and allowed to dry overnight under sterile conditions in a laminar flow box. Before use, samples were rinsed three times with phosphate buffer solution (PBS). RGD Peptides: Arginine-glycine-aspartic acid (RGD) peptide immobilization was conducted by surface modification with 3aminopropyltriethoxysilane (APTES)/N-succinimidyl-3-(2pyridyldithio)propionate (SPDP) followed by coupling of a cysteineterminated RGD nonapeptide with the sequence C-Y-G-G-R-G-D-TP. After cleaning with steam microwave plasma the samples were coated with 100 mM APTES/toluol (ABCR) for 2 h at 60 °C and 5 mM SPDP (Bachem, Switzerland) diluted in dimethylformamide for 1.5 h at room temperature (RT). Selective coupling of the RGD nonapeptide (2 mM) was performed via thiol−disulfide exchange in 0.1 M sodium phosphate buffer (pH = 7.4) overnight at 5 °C. The peptide-grafted samples were washed thoroughly with water and dried under a stream of nitrogen. Plasma Polymerized Allylamine: Amino functionalization was performed in a low-pressure plasma process reactor V55G (plasma finish, Germany, V = 60 L) in a two-step process with the precursor allylamine, as previously reported.7 The plasma deposition time was 480 s. The elemental chemical surface composition was determined by high-resolution scanning X-ray photoelectron spectroscopy (XPS). The Axis Ultra (Kratos, UK) was run with the monochromatic Al Kα line at 1486 eV (150W) as already described.7 Carboxyl Functionalization: Maleic anhydride (Prolabo, 99.5% purity) was ground into a fine powder and loaded into a stoppered glass gas delivery tube. Plasma polymerization experiments were carried out in an electrodeless cylindrical glass reactor (6 cm diameter, 680 cm3 volume, base pressure of 5 × 10−4 mbar, and with a leak rate better than 1.0 × 10−10 kg s−1) enclosed in a Faraday cage. The chamber was fitted with a gas inlet, a Pirani pressure gauge, a two-stage rotary pump (Edwards) connected to a liquid nitrogen cold trap, and an externally wound copper coil (4 mm diameter, 5 turns). All joints were grease-free. An L-C matching network (Dressler, VM 1500 WICP) was used to match the output impedance of a 13.56 MHz radio frequency (R.F.) power supply (Dressler, Cesar 133) to the partially ionized gas load by minimizing the standing wave ratio of the transmitted power. During electrical pulsing, the pulse shape was monitored with an oscilloscope, and the average power ⟨P⟩ delivered to the system was calculated using the following expression: ⟨P⟩ = Pp [ton/(ton + toff)], where Pp is the average continuous wave power output and ton/(ton + toff) is defined as the duty cycle. Prior to each experiment, the reactor was cleaned by scrubbing with detergent, rinsing in propan-2-ol, and oven drying, followed by a 30 min highpower (60 W) air plasma treatment. The system was then vented to air, and the substrates were placed in the center of the chamber, followed by evacuation back down to base pressure. Subsequently, maleic anhydride vapor was introduced into the reaction chamber at a constant pressure of 0.2 mbar and at a flow rate of approximately 1.6 × 10462
DOI: 10.1021/acsami.6b16430 ACS Appl. Mater. Interfaces 2017, 9, 10461−10471
Research Article
ACS Applied Materials & Interfaces 10−9 kg s−1. At this stage, the plasma was ignited at 60 W and run for 3 min in continuous mode. Then, pulsed mode was used for 30 min (power output = 20 W, pulse on-time = 25 μs, off-time = 1200 μs). Upon completion of deposition, the R.F. generator was switched off, and the monomer feed was allowed to continue flowing through the system for a further 2 min prior to venting up to atmospheric pressure. In contact with ambient humidity, hydrolysis gave rise to the formation of acid groups by opening the anhydride cycles. Methyl Functionalization: The maleic anhydride plasma polymer deposited films were reacted with propylamine (Aldrich, 99+%) for 1 h. This was carried out under a vacuum without exposure to air. Upon termination of exposure, the propylamine reservoir was isolated, and the whole apparatus pumped back down to the system base pressure. Then the methyl-functionalized surface was removed from the reactor and placed into an oven at 120 °C for 1 h.16 Atmospheric Plasma: This surface functionalization was performed by treatment of the sample surface with the atmospheric plasma jet kINPen08 (INP Greifswald, Germany) with an input power of 2−3 W.17 A high frequency voltage of 1.82 MHz at 2−6 kV was coupled to a centered needle electrode. Argon (99% AlphaGaz, Air Liquide, Germany) combined with 1% oxygen (medical oxygen, 99.5%, Praxair Deutschland, Germany) was used as a carrier gas with a gas flow of 5 slm (standard liter per minute) controlled by a flow controller (MKS Instruments, Germany). The length of the visible plasma plume was 7 mm. The sample was adjusted on a navigable desk, and the treatment was performed using a meandering pattern for 1 min total treatment time, whereby the plasma tip had contact with the sample. The measurements of this modification were performed not later than 24 h after preparation. 2.3. Water Contact Angle. The water contact angle (WCA) was measured by the sessile drop method using OCA 15EC (Data Physics Instruments, Germany) and distilled water (0.5 μL, Carl Roth, Germany). Drop images were acquired with the digital camera of the OCA, and the contact angles were assessed with the attached software (SCA 20, V.4.1.11 build 1018) (n = 5). 2.4. Atomic Force Microscopy (AFM) and Roughness Measurements. AFM measurements were carried out with a JPK Nanowizard II AFM (JPK Instruments, Germany) in intermittent contact mode (air) with a scan rate of 1.0 Hz using NCH-10 cantilevers (doped silicon, 42 N/m force constant; NanoWorld, Switzerland). Roughness statistics were derived from a 1 × 1 μm topographic AFM image (n = 3). The roughness average (Ra) value was determined from the mean of three 2D measurements and was calculated from 2D profiles that were obtained from the 3D scan. Roughness data analysis and visualization were done with the Gwyddion freeware (version 2.37, GNU General Public License). 2.5. Layer Thickness. The layer thickness of the coatings was measured with a multiple angle null ellipsometer (Multiscop; Optrel Gbr, Kleinmachnow, Germany) in a PCSA configuration (polarizercompensator-sample-analyzer). Using an He−Ne laser (power 4 mW; wavelength λ = 633 nm) as a light source, the measured quantities are the ellipsometric angles Ψ and Δ, which correspond to the changes in amplitude and phase of the light due to reflection at the sample. The ratio of the reflection coefficients rp and rs of the parallel and normal components of the electric vector are related to the ellipsometric angles (with respect to the plane of incidence). The structure of the sample perpendicular to the surface is represented by a stack of four slabs, each with a constant refractive index (Ti substrate, TiO2, coating, and ambient air). The refractive indices in this model were set and fixed to 2.14−2.92i, 2.59, 1.5, and 1, respectively.18,19 The thickness of the TiO2 layer was determined on a bare Ti substrate and measures 0.9 nm. The effects of surface roughness were neglected. The resulting layer thickness was determined by a least mean squares algorithm. The ellipsometric angles of each sample were obtained at multiple angles of incidence: Measurements were carried out within a range of 52° to 80° in 2° steps (with respect to the surface normal) in both accessible ellipsometric zones. 2.6. Surface Charge. Zeta potential measurements were performed on unstructured samples (2 × 1 cm) using the SurPASS system (Anton Paar, Ostfildern, Germany) to gain information on the
surface charge. The measurements were performed in a 0.001 mol/L KCl solution ranging from pH 6.0 to 8.0 with a gap height of 100 μm. The streaming current was determined depending on the pressure (max. 400 mbar). Finally, the zeta potential was calculated according to the method of Helmholtz-Smoluchowski. Measurements were performed in quadruplicate on three independent samples. To avoid any influence of pH we repeated the measurements starting with pH 8.0 and titrated down to pH 6.0. 2.7. Cell Culture. All experiments were performed with the human osteoblast-like cell line MG-63 (ATCC, CRL-1427, LGC Promochem, Germany). In general, the cells were cultured in 75 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Germany) containing 10% FCS (Biochrom FCS Superior, Merck, Germany) and 1% gentamicin (Ratiopharm, Germany) at 37 °C in a humidified atmosphere with 5% CO2. To avoid masking of the chemical functionalities, serum-free medium was used for all experiments. Before use, the titanium arrays were washed three times in phosphate-buffered saline without Ca2+ and Mg2+ (PBS) (PAA Laboratories, Austria) and placed into 4-well NUNC dishes (Thermo Fisher Scientific, NUNC, Germany). 2.8. Immunofluorescence Staining. MG-63 cells (1.5 × 104 cells) were cultured on the titanium arrays for 24 h and then fixed with 4% paraformaldehyde (PFA, 10 min at room temperature (RT)) (Sigma-Aldrich). After washing three times in PBS, the cells were permeabilized with 0.1% Triton X-100 (10 min, RT) (Merck, Germany), washed again, and blocked with 2% bovine serum albumin (BSA) in PBS (30 min, RT) (Carl Roth, Germany). For actin cytoskeleton staining, the cells were incubated with phalloidin-TRITC (5 μg/mL diluted in PBS, Sigma-Aldrich) for 60 min in the dark, washed three times with PBS, and embedded as described below. Tubulin staining was performed by using the mouse monoclonal antiα-tubulin antibody (Santa Cruz Biotechnologies Inc., USA) (1:50). The cells were incubated with the primary antibody for 60 min at RT. After washing three times with PBS, the cells were treated with antimouse-IgG-Alexa Fluor 488 (dilution 1:200) (Invitrogen, Germany) for 30 min at RT in the dark. The samples were embedded with Fluoroshield Mounting Media (Sigma-Aldrich). The examinations were performed on an inverted confocal laser scanning microscope LSM 780 (Carl Zeiss, Germany) equipped with an argon ion laser and the He−Ne ion laser. The ZEISS oil immersion objective (C-Apochromat 63) and the ZEN 2011 (black version) software (Carl Zeiss) were used for image acquisition. Confocal microscopy with three-dimensional (3D) z-stack generation was applied to visualize signals of structures not limited to one horizontal plain of the microtopography, such as the groove plateau. Z-stacking was used to generate a 3D representation and to understand the overall cell; thus a false interpretation due to different observation levels (confocal principle) could be avoided. For the actin staining, the experiments were repeated three times, and for the α-tubulin staining the experiments were repeated twice. 2.9. Cell Elongation and Alignment. After 24 h of cell cultivation on the samples, the cells were washed three times with PBS, fixed with 4% PFA (Sigma-Aldrich) for 10 min, permeabilized with 0.1% Triton X-100 (Merck) for 10 min, and blocked with 2% BSA (Carl Roth) in PBS for 30 min. Actin cytoskeleton staining and analyzing via LSM 780 (Carl Zeiss) were performed as described above. After washing three times with PBS, the samples were embedded in Fluoroshield Mounting Media (Sigma-Aldrich). Image acquisition was done with the scanning laser microscope LSM 780 (Carl Zeiss). Quantification of the cell shape: The cell area and perimeter as well as length and width were quantified after 24 h for n = 50 cells per sample out of n = 3 experiments measured using ImageJ software. The cell shape was quantified by calculating the circularity of each individual cell, described as circularity = 4πA/P2, where A is the cell area and P is the cell perimeter. This index describes the cell shape, taking its irregularity into consideration: a circularity with the value of 1 reflects a perfectly round cell, whereas polygonal cells display a circularity
