Article pubs.acs.org/Langmuir
Aging of Plasma-Polymerized Allylamine Nanofilms and the Maintenance of Their Cell Adhesion Capacity Birgit Finke,*,† Henrike Rebl,‡ Frank Hempel,† Jan Schaf̈ er,† Klaus Liefeith,§ Klaus-Dieter Weltmann,† and J. Barbara Nebe‡ †
Leibniz-Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Straße 2, D-17489 Greifswald, Germany Department of Cell Biology, University Medical Center Rostock, Schillingallee 69, D-18057 Rostock, Germany § Department of Biomaterials, Institute for Bioprocessing and Analytical Measurement Techniques e.V., Rosenhof, D-37308 Heilbad Heiligenstadt, Germany ‡
ABSTRACT: The long-term stability and γ-sterilisability of bioactive layers is the precondition for the application of implants. Thus, aging processes of a microwave deposited, plasma polymerized allylamine nanofilm (PPAAm) with positively charged amino groups were evaluated concerning physicochemical characteristics and cell adhesion capacity over the course of one year. XPS, FT-IR, surface free energy, and water contact angle measurements elucidated not only the oxidation of the PPAAm film due to atmospheric oxygen reacting with surface free radicals but also the influence of atmospheric moisture during sample storage in ambient air. Surprisingly, within 7 days 70% of the primary amino groups are lost and mostly converted into amides. A positive zeta-potential was verified for half a year and longer. Increasing polar surface groups and a water contact angle shift from 60° to 40° are further indications of altered surface properties. Nevertheless, MG-63 human osteoblastic cells adhered and spread out considerably on aged and additionally γ-sterilized PPAAm layers deposited on polished titanium alloys (Ti-6Al-4V_P). These cell-relevant characteristics were highly significant over the whole period of one year and may not be related to the existence of primary amino groups. Rather, the oxidation products, the chemical amide group, that is, seem to support the attachment of osteoblasts at all times up to one year. coupling possibilities.13 Gas discharge plasma techniques are very well suited to this purpose. Plasma processes can not only be used for the enhancement of bonding locations (hydroxyl groups), but also for the deposition of polymeric-like thin coatings on metals with the desired reactive functional groups like amino- or carboxyl groups on the surface.1,7 Simultaneously, a greater versatility for binding biomolecules using different immobilization chemistries via various linkers is possible.7,14,15 Klee et al.16 modified titanium by means of chemical vapor deposition (CVD)− polymerization of amino functionalized (2,2) paracyclophane. Friedrich et al.17 reported on polymer surface modifications with allylamine under chemical structure retention of the monomer and a relatively high amino group density (NH2/C = 18%) during pulsed plasma polymerization by radio frequency (RF) excitation. Kylian et al.18 followed a new strategy by depositing amino-rich thin films (NH2/C = 18%) using RF magnetron sputtering of nylon-6.6 in Ar/N2 or N 2/H2 discharges. A possible problem of plasma polymers is swelling and dissolution of unbound material in aqueous environ-
1. INTRODUCTION Titanium and its alloys serve as the material of choice for loadbearing, bone-contacting orthopedic and dental implants. To tailor interactions of the bone−titanium interface, a lot of topographical and chemical strategies are under experimental observation.1−3 In particular, biochemical surface modifications with biomimetic motifs promote biocompatibility, cell physiology, and the bonding of implants with the surrounding bone.4−6 The immediate occupation of an implant surface by osteoblasts is vitally important for the application in the host. A key step in this process is the cell attachment via adhesion receptors, named integrins. Therefore, immobilized proteins and peptides similar to the extracellular matrix (ECM; e.g., type I collagen, RGD peptides) that function as ligands for integrins are commonly used to design a cell attractive interface.7−10 The covalent but also the electrostatic bonding of biomolecules to the implant surface is the preferred approach. Conventionally, it is possible to apply the wet-chemical silanechemistry. The coupling of the amino group carrying silanes, e.g., 3-amino-propyltriethoxysilane (APTES), occurs via surface hydroxyl groups of the native titanium oxide layer.11,12 But properties of this silane-linker such as their deliquescence and their tendency to polymerize and to form island-like domains affect adherence adversely and give rise to the search for other © 2014 American Chemical Society
Received: May 21, 2014 Revised: October 27, 2014 Published: October 30, 2014 13914
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ments.17−19 But the selection of appropriate plasma parameters can lead to a good compromise between a sufficient number of functional groups and insolubility in water. But which plasma polymer is especially qualified for use as a cell adherent surface coating on titanium? Findings in cell biology concerning the net negative charge of eukaryotic cells open up new surface functionalization strategies. The matrix substance hyaluronan (HA) was found to be expressed by chondrocytes20−22 and osteoblasts14,22 and is characterized by a negative charge due to carboxyl groups of the glucuronic acid. Corresponding to the outermost oxide layer, clean titanium surfaces are slightly negatively charged.14,23 Therefore, a positively charged implant surface at physiological pH-value with NH2-groups24−26 as charge carriers should be applied to stimulate the electrostatic immobilization of HA, as our former studies involving plasma polymerized allylamine (PPAAm) pointed out.14,26−31 The following chemical equation describes the electrostatic relation between the two partners:
diameter) were used for the chemical functionalization, physicochemical analysis, and in vitro cell culture. The surface roughness (Ra) was determined with a surface profiler Dektak3ST (Veeco, USA). The radius of the standard diamond stylus was 2.5 μm, the stylus force 30 mg, and the scan length 8 mm. The measurements were carried out 10 times in different directions giving an average value of Ra = 0.19 ± 0.02 μm. 2.2. Deposition of Plasma-Polymerized Allylamine (PPAAm) Thin Films. A commercial microwave plasma reactor (2.45 GHz; V55G, Plasma Finish, Germany) was used for the deposition of PPAAm films with a thickness of about 50 nm.14 Substrates were positioned downstream, 9 cm under the coupling window. First the substrates were cleaned and activated by a continuous wave (cw) oxygen plasma (500 W, 50 Pa, 100 sccm O2/25 sccm Ar, 60 s) and, without breaking the vacuum, subsequently coated with a 50−100 nm thin layer of PPAAm using a pulsed low pressure microwave discharge plasma (500 W, 50 Pa, duty cycle DC 0.15, total pulse length 2 s, excitation pulse length 300 ms) with the monomer allylamine (H2C CHCH2NH2) (VWR International GmbH, Germany).14 A liquid handling system allowed exact dosing of allylamine by a calibrated needle valve (0.125 ± 0.009 mL/min). Argon was applied as a carrier gas (50 sccm). The samples were stored without sterilization or γsterilized (Co 60, minimal irradiation dose 25 kGy, Synergy Health Radeberg GmbH, Germany) at room temperature in closed petridishes in the dark and ambient air. The samples for all investigations were stored for 0, 1, 7, 30, 90, 180, and 360 d and for XPS analyses additionally for 600 d. 2.3. Surface Characterization. 2.3.1. X-ray Photoelectron Spectroscopy (XPS). The elemental chemical surface composition and chemical binding properties of the PPAAm thin films were determined by XPS at different time points up to 600 days. The Axis Ultra DLD electron spectrometer (Kratos, UK) runs with monochromatic Al Kα radiation (1486 eV; 150 W) and implemented charge neutralization. The spot size was 250 μm in diameter. Spectra were recorded at a pass energy of 80 eV for the determination of wide scans and chemical elemental composition and of 10 eV for highly resolved C 1s and N 1s peaks. Three spots in different positions on each sample were analyzed and averaged. In all cases the takeoff angle was 90°. Data acquisition and processing was performed with the “vision 2.1.3” software (operating software Kratos). Peak fitting was processed using the “Casa XPS” software version 2.14.dev29 (Casa Software Ltd., UK), with Gauss-Lorentz (30% Lorentz) distribution, Shirley baseline, and a fixed full width at half-maximum (fwhm) between 0.9 and 1.2 eV for the C 1s peak and 1.2−1.5 eV for the N 1s peak, respectively. The CC/CH component of the aliphatic C 1s peak was set to 285.0 eV. The other components of the C 1s peak were fixed to known values: amines (CNH) and secondary carboxyls (OCO) at 285.7 ± 0.1 eV; hydroxyls, ethers, imines, nitriles (CO, CN, CN) at 286.6 ± 0.2 eV; aldehydes, ketones, amides (CO, NCO) at 288.0 ± 0.3 eV; esters and carboxyls (CC(O)OX) at 289.2 ± 0.2 eV) and for labeled primary amino groups CF3 at 292.7 ± 0.2 eV.36,44 Chemical derivatization with trifluorobenzaldehyde (TFBA) (Alfa Aesar, Germany) at 40°C for 2 h over a saturated gas phase was used for the labeling of primary amino groups on the desired storage date. The density of the amino groups NH2/C was determined from the fluorine elemental fraction.37 2.3.2. Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRRAS). The chemical composition and molecular structure of PPAAm thin films were analyzed by means of FTIRRAS (FT-IR Type: Spectrum One, PerkinElmer, Germany).38 The IRRAS unit of the FT-IR spectrometer in the sample compartment used parallel-polarized light at an incidence angle of 75°. To improve the sensitivity of the FT-IR measurement, Au films were deposited on a glass substrate as reference and then coated with PPAAm. In this way FT-IRRAS measurements of minimal PPAAm film thicknesses (∼30 nm) were possible. The spectra were obtained at the spectral resolution of 4 cm−1, with the number of scans at 32, in the wavenumber region 4000 to 600 cm−1. 2.3.3. Surface Free Energy, Contact Angle. The polar and disperse part of surface free energy was calculated from measurements of
PPAAm coatings on titanium prepared by microwave plasma have favorable physicochemical properties. Typically they are very thin (∼50 nm), adhere well, are homogeneous, are strongly cross-linked, are resistant to delamination, are mechanically stable, and are equipped with a sufficient density of positively charged amino groups.29,14 In more comprehensive studies different cell biological aspects associated with PPAAm on titanium surfaces, like the mobility of the cytoskeletally associated protein vinculin,27 the metabolic activity of osteoblasts, and their long-time adhesion on PPAAm28 as well as the relation between topography and chemistry of PPAAm coated and uncoated titanium surfaces with diverse surface roughnesses of their adjusted topography, were examined.32 Intramuscular implantation of PPAAmcoated test samples in rats revealed a reduced inflammation reaction compared to uncoated titanium in vivo.30 For the implementation of plasma polymer films (PPAAm) as a bioactive implant surface design it is indispensable to know more about the long term characteristics of the physicochemical layer properties.33−35 The alterations of the film composition (as detected by X-ray photoelectron spectroscopy (XPS)) and the characterization of the surface functional groups (by Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS)) as well as the variation of surface free energy, water contact angle, zeta potential, and γ-sterilisability is of relevance. Therefore, the focus of this study is to identify (i) whether general compositional alterations of the PPAAm layer occur during a storage time of one year in ambient air, (ii) whether they occur after γ-sterilization, and (iii) whether both processes influence the long-time cell adhesive capacity of this nanolayer. To achieve an understanding of surface chemistry effects on cell adhesion and spreading, the chemical alterations have to be investigated using cell experiments precisely at distinct, combined time points over a span of up to 360 days.
2. MATERIALS AND METHODS 2.1. Substrates: Titanium Alloy and Reference Specimens. Polished titanium alloy disks (Ti-6Al-4V_P, cp, grade 2; 1 or 3 cm in 13915
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a thickness of ∼50 nm deposited on a Ti-6Al-4V_P surface.14 The polished Ti-6Al-4V_P substrates plus PPAAm film were very smooth with an average roughness of Ra = 0.25 ± 0.1 μm. The XPS detected only the elements carbon, nitrogen, and oxygen in the first 10 nm of the surface. The film was homogeneously distributed, and no Ti was observable.14 The elemental XPS analysis showed an N/C ratio of 27.2 ± 0.3%, which is near the theoretical N/C value (33.3%) of the precursor allylamine. The O/C ratio was 5.6 ± 0.4%, most probably due to an oxygen uptake during initial air contact immediately after the plasma process. Within one year’s time, N/C decreased by ∼8% to 15%, some nitrogen content is lost in the form of gaseous ammonia NH3, and O/C increased by ∼11% to 17%. The altered PPAAm composition, in particular the evolution of the N/C and O/C element ratios as a function of storage time, is summarized in Figure 1a. The NH2/C ratio of 2.5 ± 0.5%, determined by derivatization with TFBA after plasma deposition, was much lower than the ratio in the allylamine monomer, an indication for a highly cross-linked polymer-like network. However, the density of primary amino groups NH2/C changed drastically in the course of one year (Figure 1b). The total loss of primary amino groups in this time
contact angles with different liquids. Water, ethylene glycol, and methylene iodide contact angles were determined with the help of the contact angle measuring system OCA 30 (Data Physics Instruments GmbH, Germany) by the sessile drop method. The drop size was 0.5 μL. The software SCA20 was employed for device control and data analysis. The surface free energy was calculated using the methods of Owens et al.39,40 The measurements were performed at the latest 30 min after preparation and repeated three times. 2.3.4. Zeta-Potential. Existing surface charges can be estimated by determining the so-called zeta-potential. Streaming potential measurements were carried out for various pressures using the Electrokinetic Analyzer SurPass (A. PAAR K.G., Germany) to determine the streaming potentials dependent on the pressure. The zeta-potential was calculated according to the method of Fairbrother and Mastin.41 The analysis was performed in a 0.001 M potassium chloride (KCl) solution at pH 6. Poly(diallyldimethylammonium chloride) (PolyDadMac) was used as a reference surface with a positive zeta potential. 2.4. Cell Culture. The human osteoblast-like cell line MG-63 (ATCC, CRL-1427, LGC Promochem, Germany) was used for all cell experiments. The cells were cultured in 75 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) with 10% fetal calf serum (FCS Gold, PAA Laboratories GmbH, Austria) and 1% gentamicin (Ratiopharm GmbH, Germany) at 37 °C in a humidified atmosphere with 5% CO2. During the experiments cells were cultured serum-free on all Ti-6Al-4V_P samples to avoid any masking of the charged groups.14 2.5. Cell Adhesion. Suspended MG-63 cells in serum-free DMEM were seeded onto all Ti-6Al-4V_P samples for 5 and 10 min at a density of 1 × 105/specimen, and nonadherent cells in the supernatant were counted and analyzed by flow cytometry (FACSCalibur, BD Biosciences, Germany). For data acquisition the software CellQuest Pro 4.0.1 was used. Cell adhesion was then calculated in percent of the cell number at 0 min. 2.6. Cell Spreading. Human MG-63 osteoblasts were trypsinated and washed in phosphate buffer solution (PBS), and the cell membrane was stained with the red fluorescent linker PKH26 (PKH26 General Cell Linker Kit, Sigma-Aldrich, USA) for 5 min in suspension. The cells were then seeded onto all Ti-6Al-4V_P discs and cultured for 30 min, 60 min, and 24 h. After fixation with 4% paraformaldehyde (PFA, Merck, Germany) the cells were embedded with a coverslip. Spreading (cell area in μm2) of 40 cells/specimen was measured using the software “area measurement” of the confocal microscope LSM 410 (Carl Zeiss, Germany), equipped with an argonion laser and a 63× water objective (1.25/0.17, Carl Zeiss, Germany). The image size was 512 pixels × 512 pixels. 2.7. Scanning Electron Microscopy (SEM). The sample surfaces were investigated using an SEM DSM 960A (Carl Zeiss, Germany). For cell analyses, cells were grown on the samples for 30 min, 60 min, and 24 h, fixed with 4% glutaraldehyde (1 h), dehydrated through a graded series of acetone and dried in a critical point dryer (K 850, EMITECH Germany). Gold sputtering was performed with a coater (SCD 004, BAL-TEC, Balzers, Liechtenstein). 2.8. Statistics. Statistical analyses were performed with the software SPSS Vers. 15.0 for Windows (SPSS Inc., USA) using the Mann-Whitney U-Test and ANOVA posthoc Bonferroni test. Data were presented as mean; a probability value of p < 0.05 was considered significant.
3. RESULTS The change of the surface composition was studied by several analyzing techniques such as XPS, FT-IR, surface free energy, water contact angle, and zeta potential. These have different analytical depths in the range of the interfacial forces, e.g., XPS, surface free energy, and FT-IR, with 2−10 nm, 2−10 nm, and 200 nm−2 μm, respectively. 3.1. Physicochemical Surface Properties of PPAAm during Storage in Ambient Air. The starting point was a freshly prepared PPAAm film, deposition rate 3.1 nm/min, with
Figure 1. a, b: XPS element ratios N/C, O/C (a) and the amino group density NH2/C (b) as a function of storage time of PPAAm until 600 d. Note the theoretical composition N/C of the precursor allylamine (AAm), solid line at 33.3% (a), and the rapid decrease of primary amino groups within the first 7 days in ambient air (b). Repeated investigations (open circles) confirmed the curve progression of NH2/ C (black triangle) (b). The plotted curves serve as guide to the eye. 13916
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CO, NCO (288.0 ± 0.3 eV), and COOH bonds (289.2 ± 0.2 eV) in the first 15 days was followed by a slower progress over the time of one year reflecting the proceeding oxidation process and the reaction with air moisture. Highly resolved XPS N 1s spectra confirmed these results (Figure 2b). Different NC bonds led to a peak broadening between 397 and 402 eV and could not be distinguished from each other. Hence, this broad peak was formally divided into three parts CN (1) , CN (2) , and CNOH: CN (1) and CN (2) for miscellaneous covalent CN bonds such as CNH2, CN, CN, CNC, OCNH2, and OCNHR. After 360 storage days the intensity of the total peak is clearly reduced. The CN(1), CN(2), and CNOH peaks decrease in the same relation. Bonds at binding energies >402 eV characteristic for an oxidation of nitrogen, for instance, nitroso CNO, nitro CNO2, nitrite CONO, or nitrate CNO3 bonds, were not found.43 An oxidation of nitrogen did not take place. In addition to the figures above, Figure 3 shows an overview of the starting and end conditions in N/C and O/C and the
period was at about 90%. A relatively quick aging process entailed a loss of 70% of primary amino groups within the first 5−7 days and in a slower, subsiding process a loss of a further 20% within the following half year. The same curve progression could be reconfirmed by repeating the investigation (open circles in Figure 1b). The plotted curves serve as guide to the eye. The equations of the curves in Figure 1a,b were obtained by fitting the data with exponential functions under the assumption of first order kinetics. From these, the time constants t were calculated. Those values are very different: for N/C ∼110 days, O/C ∼30 days, and NH2/C 2−5 days. This means that the decay and uptake processes seem to be very complex with few correlations. Not only oxidation reactions of primary amines but also the inclusions of free radicals, which occur in parallel, have to be kept in mind.42 Because of this, the single exponential fit is a rather crude approximation. High resolution XPS C 1s spectra of PPAAm (Figure 2a) verified the existence and changes of different CC-, CH-,
Figure 3. Changes of N/C- and O/C-ratios as well as CX bonds from C 1s high resolution spectra as deposited and stored for one year in ambient air.
fitted CX bonds of C 1s high resolution measurements of a freshly prepared and an aged PPAAm coating after 360 days. It is confirmed that primary amino groups (CF3 groups) have been lost as well as CN, CO, and CN bonds decreased, whereas amide NCO, carbonyl CO, and carboxyl OCO bonds were found to increase in this time period. FT-IRRAS studies of the PPAAm thin films immediately after deposition (0 h) and after storage in ambient air over 7, 30, 90, and 360 days, respectively, are shown in Figure 4a. In particular, the FT-IRRAS spectrum of PPAAm after deposition (0 h) confirmed a high retention of the structural properties of the monomer allylamine H2CCHCH2NH2 for the deposition method used here. Basic features of the monomer structure like the two stretching vibration bands of primary amines ν-NH at about 3500−3000 cm−1, the symmetric/ asymmetric stretching vibrations ν-CH2,3 at 2980−2880 cm−1, and the deformation vibrations of amines, δ-NH at 1650−1510 cm−1, as well as the deformation vibration δ-CH2,3 at 1465− 1375 cm−1 were still dominant in the spectrum.44−46 But typical plasma effects were also observable, e.g., significantly
Figure 2. a, b: High resolution XPS analysis of the C 1s (a) and N 1s (b) spectra of PPAAm at different time points up to 360 d in ambient air. Note (a) the decreasing primary amino groups (CF3 bonds near 297.7 eV) and the increasing acid amide bonds (CO, NCO at 288.0 ± 0.3 eV) and carboxylic acid bonds (COOH at 289.2 ± 0.2 eV). The N 1s peak (b) is formally fitted by different peaks which are marked by CN(1), CN(2), and CNOH. CN(1), CN(2) for miscellaneous covalent CN bonds: CNH 2 , CN, CN, CNC, OCNH 2 , OCNHR after deposition and storage in ambient air for 360 d.
nitrogen-, and also oxygen-containing bonds at a specific date; at the day of deposition and after 7, 30, and 360 days of storage in ambient air. The bond areas of CN (285.7 ± 0.1 eV) and CO, CN (286.6 ± 0.2 eV) ran through a maximum in the first 30 days (not shown here) while the primary amino groups (CF3 bonds near 292.7 eV) decreased promptly within even fewer days. The initial increase of 13917
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Figure 4. a−d: FT-IRRAS spectra for (a) PPAAm after deposition (0 h) and storage in air up to 360 days. (b) The difference between the FTIRRAS reflection spectra after deposition and after 360 days storage shows the formation and degradation of characteristic molecular groups. (c) Analysis of the IRRAS reflection band at 1750−1500 cm−1 reveals peak broadening and shifting to higher wave numbers as well as growing substructures during aging. (d) The substructures of the IRRAS reflection band (360 d) at 1750−1500 cm−1 can be assigned to ν-CO (amide I) at 1655 cm−1, ν-CN (amide II) for primary amides at 1602 cm−1, and a further ν-CN band (amide II) at 1563 cm−1 for secondary amides.
and are especially pronounced after 360 days of storage in ambient air (Figure 4c). The band minima as well as the band edge were shifted to higher wave numbers under band broadening with storage time. An analysis of the substructures was carried out. After one year of storage in ambient air, characteristic amide bands (Figure 4d) were found. For all amides, the reflection of amide I is at 1655 cm−1 (ν-CO stretching vibrations). The wavenumber of the amide II band depends on the type of amide group present. The band at 1602 cm−1 can be assigned to the amide II vibration of primary amides and the band at 1563 cm−1 to the amide II band of secondary amides. The amide III band is found at 1240 cm−1 νs-CN stretching/δ-NH bending vibrations (Figure 4a).49−51 Changes of the surface free energy and water contact angle with time were further indications for the aging process. Both measurements showed a characteristic curve progression (Figure 5a,b) which was confirmed by threefold determination on PPAAm samples prepared in different runs. The polar part and likewise the total surface free energy of the PPAAm thin film decreased about 7 mN/m in the first 7 days followed by an increase until saturation at about 37 or 57 mN/m, respectively, after half a year of storage. In parallel, the disperse part of the surface free energy went through a little initial maximum and into a diminished level at about 20 mN/m after approximately 180 days. The water contact angle rose from 47° after the preparation process to 57° in the first 7 days
broadened, disappearing, or arising bands. Amino groups of the precursor were partially transformed into amide, imine, or nitrile functional groups by the plasma deposition process. An indication for that is the new band between 2300−2100 cm−1 associated with stretching vibrations of nitrile and ethine groups, ν-CN and ν-CC,47 as well as the imine group CN and ethene group CC at 1690−1650 cm−1. The comparison of the IRRAS reflection spectrum measured directly after deposition (0 h) with that taken after storage in ambient air (360 d) exhibits significant molecular changes in the PPAAm film (Figure 4b). The difference spectrum gives important information about the formation and degradation of molecular groups and the broadening and shifting of IRRAS reflection bands. Characteristic molecular changes can be identified: the formation of OH groups due to water vapor absorption, ν-OH stretching vibrations at 3700−3000 cm−1, and carbonyl groups ν-CO stretching vibrations at 1700− 1650 cm−1, an indication for arising acid amides. The reflection at 1465−1300 cm−1 can be assigned to deformation vibrations of δ-CH and OH groups. Furthermore, CN and CC triple bond structures decreased, indicating the loss of unsaturated structures.48 The formation of acid amides over time is characterized by overlapping and peak broadening of δ-NH deformation vibration bands and ν-CO stretching vibration bands between 1750 and 1500 cm−1. In the FT-IRRAS spectra band substructures can be observed in this range already after 7 days 13918
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Figure 6. Effect of γ-sterilization on surface free energy of PPAAm (unsterile and γ-sterilized) at days 7 and 360. Note the relatively low influence of the γ-sterilization at day 7 and the somewhat higher at day 360.
3.2. Cell Adhesion Capacity during the Aging Process of PPAAm Thin Films. Initial cell adhesion experiments revealed a significant increase of adherent cells on the PPAAmmodified Ti-6Al-4V_P surface. A more than 2.3-fold increase compared to untreated Ti-6Al-4V_P was measured for all samples at every time point within the first week of storage. Although investigations were performed under standardized cell culture conditions, e.g., the use of cell passages between 5 and 25, variations in the control have occurred over the long period of one year. However, it is clearly visible that cell adhesion on PPAAm is consistently increased at all time points and the results show cell adhesion of 88.4−95.9% and 80.1− 96.4% after 5 and 10 min, respectively (Figure 7a). Furthermore, it was found that the surface modification with PPAAm boosts the spreading (30 min, 60 min, 24 h) of osteoblasts (Figure 7b). This significant increase was detected for all tests over the whole period of one year. It was even more pronounced in the cell spreading phase at the time point 30 min, where a nearly 2.2-fold increase was observed at day 0. After 24 h the PPAAm layer led to a 1.6-fold increased cell area compared to untreated Ti-6Al-4V_P. This increase can also be seen at day 180: 2.0-fold after 30 min and 1.6-fold after 24 h. Even after 360 days of storage the PPAAm layer can boost the 30 min as well as 24 h cell spreading by a factor of 1.8 and 1.6, respectively. The cell area is still visibly larger at all time points after 360 days of storage. These results underline the superior effect of PPAAm in the first hours of cell attachment and spreading and also demonstrate the positive effect of PPAAm modification that is stable throughout a one year period. Analyses of the cell morphology demonstrate that the cells on PPAAm-modified Ti-6Al-4V_P spread on the surface already after a contact time of only 30 min (Figure 8). Cells on the uncoated Ti-6Al-4V_P surface appear rather rounded and only loosely bound with few filopodia. Overall, on PPAAm the cell area is highly increased, resulting in an outstanding cellsurface contact even on a PPAAm surface stored for 360 d. The differences in short time (30, 60 min) and long term spreading (24 h) between unsterile and γ-sterilized PPAAm surfaces on days 7 and 360 are minimal (Figure 9a,b). SEM images (Figure 10) confirm this observation. Thus, this
Figure 5. a, b: (a) Surface free energy of PPAAm with its polar and disperse parts determined up to 360 days (three measuring points/ sample, n = 3 independent experiments until 200 days). Note the decrease of polar groups and total surface free energy in the first 7 days followed by a further increase. (b) Water contact angle measurements of PPAAm determined up to 360 days (one measuring point/sample, n = 3 independent experiments until 200 days). Note the increase of the water contact angle in the first 7 days followed by a subsequent decrease in the WCA.
of storage and decreased subsequently to 37° by a rising number of polar hydrophilic groups in the first half of the year. The influence of γ-sterilization on the PPAAm film is relatively low (Figure 6). The measurement of the surface free energy and water contact angle (not shown) of the unsterile PPAAm surface was carried out immediately after return of the γ-sterilized samples at about the seventh day. Therefore, both starting contact angles are comparatively high at about 60° (compare Figure 5b). The difference between the unsterile and sterile PPAAm surface at day 360 is higher. More polar groups and thus a lower water contact angle are found at the sterile surface. Investigations of the zeta potential confirmed a positive surface charge of the PPAAm surface during storage in ambient air for a half year (2 days: 13.9 ± 1.2 mV; 20 days: 20.9 ± 4.8 mV; 200 days: 26.3 ± 0.5 mV). This slightly increasing positive zeta potential was unexpected first since the density of primary amino groups decreased very quickly in the first 7 days. But other positively charged N-functional groups such as imines, nitriles, and acid amides contribute as well to the overall surface charge in a relatively long time span of more than half a year. Negatively charged functional groups seem to have only little influence in the first time. A small negative surface charge of −17 mV could be determined after storage of 2 years and longer. 13919
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sterilization method is well suited for PPAAm and does not further alter the chemical characteristics of PPAAm.
4. DISCUSSION The reason for the progressive aging processes of deposited plasma polymers is based on the physics and chemistry of the plasma process itself. In general, plasma polymerization processes at low pressure are due to radical reactions on the surface. Neutral transient reactive species from precursor fragmentation in the plasma volume are adsorbed on the surface and polymerize. Plasma−surface interactions and VUV/ UV photons contribute to the film growth.52 Accordingly, the plasma polymerization is a plasma-initiated and -supported process leading to a random hydrocarbon network of amorphous organic thin films with functional groups from the precursor molecule and amounts of free radicals remaining in the film and on the surface, bound to carbon or motile. Especially tertiary carbon radicals are long-lasting, living for many weeks and therefore stable for a long time. 53 Furthermore, the use of pulsed plasmas in turn enhances the pure chemical chain propagation, resulting in chemically betterdefined polymer structures. The radical chain reaction is terminated by disproportionation, a radical−radical recombination, or a reaction with oxygen molecules from the rest of the gas. Therefore, pulsed plasmas with long plasma-off periods and short plasma pulses offer a good compromise to produce chemically formed polymer structures with a minimum of irregularities and high chemical structure retention.44,54 After finishing the plasma process and sample transfer in ambient air, the functionalized polymer surface is promptly overlaid by the reaction of plasma-generated free radicals with oxygen from air, by the so-called post-plasma oxidation or autooxidation processes.55 The initial bound carbon radicals react with atmospheric oxygen to peroxy-radicals. Different intermediary and end products such as carbonyls, carboxyls, or even new carbon radicals can be formed in a series of reactions.55−57 Such processes are kinetically favored and, thus, very fast, because the oxygen molecule can be also considered as a biradical.53 Furthermore, it is assumed that motile radicals localize at the carbon atom anchor of the electron-rich primary amino group and promote their oxidation to acid amide groups and partially to imides.57 Figures 1b and 2a show the rapid conversion of primary amino groups into acid amides within 5− 7 days after deposition. XPS and FT-IR confirm both the formation of acid amides and carboxylic groups. An oxidation of nitrogen could not be found (Figure 2b). Stable oximes can also be detected. But also hydrolysis takes place during the storage of PPAAm in ambient air. Atmospheric moisture influences reactions of the existing and arising surface functional groups (possible reaction pathways in Figure 11a,b). Imines react to ketones or aldehydes plus ammonia (nitrogen loss) and nitriles react to acid amides. In contrast, acid amides are hydrolyzed to carboxylic acid groups and ammonia only under very harsh reaction conditions with concentrated acids or bases at temperatures over 100 °C. For this reason this reaction pathway seems to be irrelevant in our case. The nitrogen loss remains limited (Figure 1); only imines seem to contribute (Figures 3, 11a). CO, NCO, and COOH bonds increase slowly during the storage in ambient air in the course of one year (Figures 2a and 3). Acid amides are available in sufficient amounts during one year storage (Figures 2a, 3, and 4b−d), as physicochemical analysis indicates, displaying, e.g., a
Figure 7. (a) Initial cell adhesion after 5 and 10 min is significantly enhanced on Ti-6Al-4V_P surfaces biofunctionalized with PPAAm. Note the consistently good cell adhesive property of the plasma polymer film over the whole period of one year/360 days (n = 4 independent experiments, Mann-Whitney-U-Test, *p < 0.05). (b) Short time (30, 60 min) and long-term spreading (24 h) of MG-63 cells on PPAAm-modified surfaces. Note that the cell area is significantly enhanced over the whole period of 360 days (n = 40 cells, ANOVA posthoc Bonferroni ***p < 0.005; **p < 0.01). 13920
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Figure 8. Morphology (SEM) of MG-63 cells (after 30 min) on untreated Ti-6Al-4V_P (left) and a PPAAm surface (right), both stored for 360 d. Cells on the untreated Ti-6Al-4V_P are attached to the surface with only a few filopodes. In contrast, it is clearly visible that the cells spread far better on the PPAAm-samples, even though the samples had been stored for one year. The cell area is highly increased, resulting in outstanding cell− surface contact (magnification 2000×, 45°, DSM 960A, Carl Zeiss; bar = 10 μm).
oxidation productthe chemical amide groupobviously supports the attachment of osteoblasts at any time within the one-year period. The first seven days of storage in ambient air are very important for the PPAAm surface alteration. Not only were a decrease of polar surface groups and an increasing water contact angle and thus more hydrophobic surface properties observed in this time period, but also a rapid loss of primary amino groups and a change of the functionalities were observed. The reason for the increasing hydrophobicity during the initial phase of aging was explained by van Os58 and Griesser et al.;59 they cited the reorganization and restructuring of the PPAAm surface as the dominating process. The hydrophilic amino groups move away at a certain degree from the surface into the bulk, leading to a more hydrophobic surface with a higher density of hydrocarbon segments. Different processes, e.g., oxidation and hydrolysis, contribute in the first few days to the overall aging process (Figures 1 and 11). The formation of a higher fraction of polar groups by further oxidation leads to more hydrophilic surface properties with a lower water contact angle. The short time spreading for 30 and 60 min of MG-63 cells (Figure 7b) seems to be affected in the first 7 days by these PPAAm surface alterations. However, the values of the 24 h spreading leveled and the adhesion experiments also showed no influence of this effect. Nevertheless, the existing nitrogen functional groups can still counterbalance the negatively charged oxygen functional moieties of the surface. Zeta potential investigations confirmed a positive surface charge in the first half of the year and longer. Cell culture investigations confirmed the predominance of PPAAm thin film coated surfaces over the whole time compared to pure Ti-6Al-4V_P. Our experiments revealed that an additional sterilization process of PPAAm with γirradiation, necessary for medical applications, did not influence the improved spreading behavior of osteoblasts. It could be shown that MG-63 human osteoblastic cells adhered and spread out considerably also on aged and γ-sterilized PPAAm layers deposited on Ti-6Al-4V_P. The improvement of the cell adhesion capacity of PPAAm in comparison to Ti-6Al-4V_P could be preserved over the one-year period. Therefore, PPAAm thin films are very well-suitable for the finishing of cell adhesive implant surfaces.
Figure 9. Short time (30, 60 min) and long-term spreading (24 h) of MG-63 cells on untreated Ti-6Al-4V_P and PPAAm treated Ti-6Al-4V_P, unsterile and γ-sterilized, after about 7 days (a) and 360 days (b) of storage in ambient air. Note that the cell area is significantly enhanced over the whole period of 360 days and not influenced by the sterilization process (n = 40 cells, ANOVA posthoc Bonferroni ***p < 0.0001).
decrease and conversion of nitriles into acid amides in the FTIR spectra (Figures 4a,b and 11b). Chemically unstable functional groups are converted into stable amides and also oximes. These findings are consistent with conclusions reached earlier by Griesser et al.57 The zeta-potential is positive for half a year and longer. MG-63 human osteoblastic cells show considerably enhanced adhesion and spreading also on these “aged” PPAAm films, compared with untreated Ti-6Al-4V_P. These cell-relevant characteristics are highly significant over the whole period of one year and do not seem to be related to the existence of primary amino groups alone. Instead, their 13921
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Figure 10. Morphology (SEM) of MG-63 cells (24 h culture) on unsterile and γ-sterilized PPAAm surfaces after 360 days of storage. The cell area is still highly increased resulting in an outstanding cell−surface contact (magnification 1000×, DSM 960A, Carl Zeiss, bar = 20 μm).
PPAAm thin films it was important to demonstrate that the outstanding cell−surface contact of MG-63 osteoblasts (adhesion and spreading) was maintained even after storage of the PPAAm modified Ti-6Al-4V_P samples for up to one year and after γ-sterilization.
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AUTHOR INFORMATION
Corresponding Author
*(B.F.) Tel.: +49 3834 554433. Fax: +49 3834 554301. E-mail: fi
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Federal Ministry of Education and Research (Grants 13N9779, 13N11183, and 13N11188, Campus PlasmaMed) as well as by the program TEAM of Mecklenburg-Vorpommern and the Helmholtz Association in Germany (UR 0402210, VH-MV1). We appreciate the technical support of R. Ihrke, Dr. A. Quade, U. Kellner, G. Friedrichs, U. Lindemann, D. Jasinski, and A. Schella from INP Greifswald, S. Staehlke from the University of Rostock, and H. Rothe from IBA Heilbad Heiligenstadt.
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Figure 11. Possible reaction pathways: (a) hydrolysis of imines to carbonyl functional groups (ketones, aldehydes) and ammonia; (b) hydrolysis of nitriles to acid amides, see ref 60.
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