Multifunctional Plasma-Polymerized Film: Toward Better Anticorrosion

Article pubs.acs.org/journal/abseba

Multifunctional Plasma-Polymerized Film: Toward Better Anticorrosion Property, Enhanced Cellular Growth Ability, and Attenuated Inflammatory and Histological Responses Pengkai Qi,†,‡ Ying Yang,†,‡ Kaiqin Xiong,†,‡ Juan Wang,†,‡ Qiufen Tu,‡,§ Zhilu Yang,*,†,‡ Jin Wang,†,‡ Junying Chen,†,‡ and Nan Huang*,†,‡ †

Key Laboratory of Advanced Technology for Materials of Education Ministry, ‡The Institute of Biomaterials and Surface Engineering, School of Materials Science and Engineering, and §Laboratory of Biosensing and MicroMechatronics, Southwest Jiaotong University, Chengdu 610031, China S Supporting Information *

ABSTRACT: Over the past few decades, plasma surface modification technique has been widely used to selectively improve surface properties and biocompatibility of materials. In this paper, at first a simple and effective method for the deposition of plasma-polymerized allylamine films onto 316L stainless steel (SS) from an allylamine/nitrogen gas mixture was developed. These amine-rich films were characterized by grazing incidence attenuated total reflection Fourier transform infrared spectroscopy (GATR-FTIR) and X-ray photoelectron spectroscopy (XPS), and the anticorrosion properties were demonstrated by electrochemical analysis. Micro-BCA and quartz crystal microbalance with dissipation (QCM-D) results showed that the higher density of amine groups of the allylamine-nitrogen plasma-polymerized film contributes to more serum protein adsorption which may enhance the adhesion and growth of cells on biomaterials. The in vitro and in vivo anti-inflammatory evaluation was performed and it has been confirmed that these nitrogen-rich surfaces could inhibit the activation of macrophages by down-regulation of the pro-inflammatory cytokines TNF-α and IL-6, and exhibit acceptable tissue-compatibility. It was found that with the help of nitrogen, plasmapolymerized allylamine films presented superior biological properties and provided a high potential application in surface modification of biomedical substrate with desirable clinical performance. KEYWORDS: surface modification, plasma polymerization, amine surface, biocompatibility, medical devices

1. INTRODUCTION Surface engineering methods have been widely used to develop artificial materials and devices that behave in human body and biological applications to repair and replace damaged or injured tissues ranging from hard tissue implants (artificial hip joints or screw-shaped tooth implant) to cardiovascular implants (vascular stents, grafts, or artificial heart valves).1−3 It has been deeply recognized that the interfacial chemical-physical properties can modulate the behavior of cells and tissue responses and thus improve the performance of biomaterials and devices.4,5 Numerous strategies have been explored to tailor the surface functionalities of the biomedical devices and enhance their biocompatibility. For instance, chemical acid or alkaline treatment,6,7 coating substrates with antifouling or biocompatible films,8,9 and ultraviolet or laser-induced patterned surfaces10,11 have all been paid much attention. Besides, plasma surface modification is an effective and economical surface treatment technique for many materials.12 It can meet complex requirements by varying plasma modification equipment including plasma sputtering, etching, © XXXX American Chemical Society

cleaning, implantation, and deposition such as preparing superhard diamond like carbon coating by plasma deposition13 or anticoagulant inorganic Ti−O films by plasma immersion ion implantation.14 Among various common plasma techniques, plasma polymerization is a unique dry process to produce ultrathin, pinholefree polymer-like layers as well as to convert monomer functional groups (especially amine,15 carboxyl,16 alkyne17 aldehyde,18 or coexistence of multiple functional groups19,20) into a polymerized molecule backbone serving as anchoring points or improve adhesive interactions. However, nitrogenrich films are the most widely deposited ones. One of the intentions is that nitrogen-rich films can promote cellular adhesion21,22 and influence differentiation of mesenchymal stem cells.23,24 The second objective is utilizing chemical active amine groups to conjugate biomolecules (such as small Received: December 11, 2014 Accepted: June 10, 2015

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DOI: 10.1021/ab5001595 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering molecules,25 proteins,26 antibodies,27 and DNA28) in mild aqueous environment. Generally, more amine groups are considered beneficial for the growth of cells and the immobilization of biomolecules, which have been widely reported.29 However, few research groups focus on this plasma-polymerized platform and give a systematical evaluation about its cellular and tissue biocompatibility. In this work, two types of nitrogen-rich platform films are taken as examples for the evaluation of the subsequent biocompatibility. The comparison of the two nitrogen-rich film families, between pulsed plasma-polymerized allylamine film (PPAam) and nitrogen-assisted deposition of PPAam film (PPAam-N2) was conducted by surface energy, atomic force microscopy (AFM), GATR-FTIR, XPS, and electrochemical tests from a perspective of material science. Besides that, we investigated the adsorption of bovine serum albumin (BSA) and multicomponent cell culture media with serum proteins, and the growth behavior of human umbilical vein endothelial cells (ECs) and osteoblasts (OBs), which play the key roles in cardiovascular or hard-tissue regeneration, respectively. By incubation of macrophages with two types of nitrogen-rich films and animal tests, the inflammatory and histological responses of these nitrogen-rich films to the body as well as their potential applications in biomedical devices are systematically evaluated and discussed.

instrument AG) by tapping model. The thicknesses of the deposited films were measured by a spectroscopic ellipsometer (M-2000 V, J.A. Woollam, USA). Δ and Ψ values were measured at a wavelength of 370−1000 nm were chosen for data analysis, and Cauchy model was used to determine the thickness of the deposited polymer layers. GATR-FTIR (Nicolet model 5700) measurement in the range of 4000− 400 cm−1 was used to analyze the chemical structure of the specimens. XPS (PerkinElmer 16PC) was applied to determine the element composition of these two families of nitrogen-rich films. The instrument was equipped with a monochromatic Al Kα (1486.6 eV photons) X-ray source operated at 12 kV × 15 mA at a pressure of 2 × 10−7 Pa. 2.4. Quantification of Amine Groups by Acid Orange II. For the quantification of surface amine (including primary and secondary amines) densities of the PPAam and PPAam-N2 films, we spread out 500 mmol/L acid orange II (AO II) solution dissolved in hydrochloric acid of pH 3 onto each group with 4 parallel samples. After interaction for 12 h at 37 °C, the specimens were washed (3 × 15 min) with hydrochloric acid solution (pH 3) to remove the weakly adsorbed AO II. Afterward, the samples were shaken for 15 min at 37 °C in NaOH solution (pH 12) to dissolve the adsorbed AO II on the surface of the samples. The AO II concentrations (which reflect the surface amine concentration of the films) of the solution was colorimetrically determined with a microplate reader (μQuant, Biotek instruments Inc.) at 485 nm. 2.5. Electrochemical Corrosion Tests. Electrochemical corrosion tests were conducted on an electrochemical workstation (IM6, Zahner, Germany) with a three-electrode setup, i.e. Specimen acted as a working electrode, a platinum sheet as a counter-electrode and a saturated calomel electrode (SCE) as a reference electrode. Fifty milliliter of PBS incubated in a water bath at 37 ± 0.2 °C served as electrolyte. The samples were sealed by silicon rubber with an uncovered working surface area of 0.75 cm2. Potentiodynamic polarization curves were scanned from −1 to 1 V at a scan rate of 2 mV/s. The natural corrosion current (Icorr) and natural corrosion potential (Ecorr) were estimated by the Tafel method. The electrochemical impedance spectroscopy (EIS) measurement shown by Nyquist plots was done in the same setup, in a scanning frequency range of 40 kHz to 0.01 Hz with a 10 mV amplitude sine wave, by a single AC mode of amplitude of 10 mV. 2.6. Protein Adsorption via Micro-BCA Assays and QCM-D Measurement. Protein adsorption was assessed using bovine serum albumin (BSA) and fetal bovine serum (FBS). The 316L SS, PPAam and PPAam-N2 groups with 4 parallel samples were immersed in the BSA (10 mg BSA in 1 mL 0.9 wt % NaCl solution) or 15% FBS solution for 1 h at 37 °C, simulating the human physiological environment. Subsequently, the samples were taken out and rinsed with PBS twice to remove the loosely adsorbed proteins. After N2 drying, all the samples were immersed in 300 μL Micro-BCA solution (A/B/C/NaCl buffer = 25:24:1:50) according to the instruction of Micro-BCA Protein Assay Kit (Pierce/Thermo Scientific, Rockford, IL, USA) and incubated for 90 min at 37 °C. The concentration of adsorbed proteins was colorimetrically determined with a microplate reader at 562 nm. To confirm the dynamic adsorption process of FBS, we used the QSense E4 system (Q-Sense AB, Sweden) to monitor the dynamic adsorption behavior of FBS on PPAam and PPAam-N2 samples in realtime. Briefly, the PPAam and PPAam-N2 films were deposited onto the gold-coated quartz crystal (diameter: 10 mm). The modified quartz crystals were settled in the QCM-D equipment chamber and Dulbecco’ modified Eagle’s medium (DMEM/F12) solution was injected continuously at a rate of 50 μL/min until the QCM traces remained steady. Subsequently, FBS solution was injected until equilibrium. After QCM running for about 1 h, DMEM/F12 solution was injected to remove the weakly absorbed proteins. The calculation method of the mass of protein adsorbed on the surface is shown elsewhere.30

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. 316L SS discs (Φ = 10 mm), the most widely used biomedical material were from New Material Co. Ltd. (Xi’ an, China). Allylamine monomer used in this study was purchased from Sigma (Purity ≥99.0%). All the other reagents used in the experiments were of the highest analytical purity (>99%). 2.2. Preparation of Plasma-Polymerized Films. PPAam films were deposited on mirror-polished 316L SS using capacitative plasma excited by an external coil with a 13.56 MHz pulsed radio frequency (RF) excitation. Argon (Ar) was used as the discharge gas, and the mixed gas of allylamine vapor and N2 was used as precursor gas. Prior to deposition, the 316L SS substrates were cleaned by Ar plasma sputtering for 5 min under the discharge power of 80W. After checking up the base pressure kept in 1.0 Pa, a flow rate of Ar (2.5 sccm) and a partial pressure of the monomer gas (3.5 Pa) were introduced into the reactor via a needle valve and maintained constant for all of the experiments. The experiments were conducted at 30 W discharge power under the condition of the pulsed duty cycle of 40% (ton = 20 ms, toff = 30 ms) and the bias voltage −80 V. As for PPAam-N2 films, an increasing nitrogen flow of 3, 4, 5, and 6 standard-state cubic centimeter per minute (sccm) was extra added to form allylaminenitrogen species atmosphere to prepare a series of PPAam-N2 films. After deposition for 45 min, the as-deposited PPAam and PPAam-N2 series films were both thermally treated at 120 °C under 1.5 × 10−4 Pa for 1 h. The detailed deposition parameters were listed in Table S1. If not otherwise specified, PPAam-N2 film refer to the nitrogen flow of 5 sccm. 2.3. Surface Characterization. Surface contact angles were measured by a drop shape analysis system DSA100 (Krüss, Hamburg, Germany) at room temperature followed by image processing of a sessile drop of 5 μL of test distilled water and diiodomethane (CH2I2) with DSA 1.8 software. The Fowkes theory was applied (eq 1) to

calculate the polar (γp) and dispersive (γd) components of the surface energy for each surface: γl and γs were related to the liquid surface tension and solid surface energy, respectively. γl(cos θ + 1) = 2 γs p γl p +

γs d γl d

(1)

The changes of the surface morphology and roughness of different samples were measured by AFM (NanoWizarII, JPK B


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Figure 1. (A) AFM images, (B) water contact angle, and (C) surface energy of the Si or SS as control, PPAam, and PPAam-N2 samples. performed to find significant differences between pairs. Probability values less than 0.05 and 0.01 were considered statistically significant and remarkable significance, respectively. In the figures, statistically remarkable significant differences (p < 0.01) and significant differences (p < 0.05) were denoted with *** and *, respectively.

2.7. In Vitro Cellular Responses. ECs were isolated from newborn umbilical cord using enzymatic digestion according to previous studies.30 Following isolation, ECs were cultured in DMEM/ F12 supplemented with 20% FBS. Primary mouse OBs were obtained from mouse calvaria and cultured in Minimum Essential Medium Alpha (α-MEM) supplemented with 5% FBS. All the cells were cultured in humidified air containing 5% CO2 at 37 °C. ECs and OBs were seeded onto SS, PPAam and PPAam-N2 samples at a density of 5 × 104 cells/mL in 1 mL cell solution. The samples after incubation for 2 h (only for ECs), 1 and 3 days were fixed in 2.5% glutaraldehyde solution followed by staining with DAPI and Rhodamine123, and then immediately inspected under a Leica DMRX fluorescence microscope (DMRX, Leica, Germany). The number of cells was determined with software assisted image analysis (Image Pro Plus and ImageJ) to evaluate the cell attachment and adhesion. The proliferation of ECs and OBs were investigated by Cell Counting Kit-8 (CCK-8) after incubation for 1 and 3 days, respectively. The medium was removed and 350 μL of fresh medium containing 10% CCK-8 reagent was subsequently added to each sample and incubated for 3 h at standard culture conditions. Afterward, the absorbance of the medium was measured at 450 nm by a microplate reader. 2.8. Anti-inflammatory Effects. Murine macrophage cells from the cell line RAW 264.7 were cultured in DMEM/F12 supplemented with 10% FBS and seeded at a density of 5 × 104 cells/mL in 1 mL cell solution. After 24 h incubation with different groups of the samples, cell culture media were centrifuged and the supernatants were harvested for further cytokines tests according to the instruction of TNF-α/IL-6 ELISA kit (Boster, China). After harvesting the cell culture medium, all the samples were rinsed by PBS and then fixed with 2.5 wt % glutaraldehyde solution. After Rhodamine123 fluorescent staining, cellular morphology was imaged under fluorescence microscope. Besides, followed by the dehydrating, dealcoholizing, and critical point drying process, the samples were sputtered with gold and the growth status of the cells was then examined by SEM. 2.9. In Vivo Animal Tests. All animal experiments were performed in accordance with protocols approved by the Local Ethical Committee and Laboratory Animal Administration Rules of China. To assess the tissue compatibility and local inflammation of an implant, we sterilized SS, PPAam, and PPAam-N2 disks (3 × 5 mm2, double surface polished and - polymerized films deposited) by ethylene epoxide and implanted them subcutaneously in the healthy New Zealand white rabbits (weight about 4 kg, n = 4). Each rabbit received two disks, placed bilaterally. After implantation for 2 and 9 weeks, the disks and surrounding tissues were excised, fixed in formalin, sectioned, mounted on slides, and stained with hematoxylin and eosin for general morphological analysis or Masson’s trichrome for analysis of collagen organization. 2.10. Statistical Analysis. All of the cell tests, AO II, Micro-BCA assay, and TNF-α/IL-6 ELISA kit, were carried out at least three times with four parallel samples. The quantitative results are reported as mean ± standard deviation (SD). All data were compared with oneway ANOVA tests to evaluate statistical significance using SPSS software. Subsequent Tukey multiple comparison tests were

3. RESULTS 3.1. Surface Characterization. The thicknesses of these two types of nitrogen-rich coatings were measured by ellipsometry. After deposition of 45 min, the thickness of PPAam was about 106.1 nm, indicating a deposition rate of 2.36 nm/min. However, the extra addition of nitrogen increased the nitrogen reactive species during plasma discharge process and consequently increased the deposition rate as shown in Table S1. As the flow rate of nitrogen increased from 3 to 6 sccm, the thickness increased at first and was reduced again latter, with a corresponding tendency of deposition rate. It is well-known that process vapor, the substrates, and the process conditions determine which process is dominant: deposition, substitution, or etching. Film deposition in monomer fragmentation-dominated plasma is dominated by complex collision and radiation processes.31 With the increase of the flux of nitrogen gas in the vacuum chamber, film growth became rapid. Once the balance of competitive ablation polymerization is broken (6 sccm nitrogen in this system), more induction of nitrogen species lead to ablation and consequently the thickness and deposition rate decreased in the case of PPAam-N2-6 sccm.32 The morphology and roughness of the surface changed. As shown in Figure 1A, the surface of the bare SS was flat with a root-mean-square roughness of 3.4 ± 0.2 nm. However, the surface roughness of the PPAam and PPAam-N2 coatings decreased to about 2.3 ± 0.3 and 2.2 ± 0.6 nm. It is confirmed that plasma polymerization is a unique technique to produce a smooth and pinhole-free surface and the introduction of nitrogen did not alter the topography of plasma modified surfaces,33 as there is no significant difference of the morphologies between the PPAam and PPAam-N2 samples. Besides, the determination of wettability and surface energy of the different samples were carried out as presented in Figure 1B. Compared with a water contact angle (WCA) of 56.2° of the PPAam, the PPAam-N2 showed a more hydrophilic property with a WCA of 46.2°. The further analysis of surface energy revealed that the polar surface energy increased from 18.13 to 25.72 mN/m adding 5 sccm N2 as assistant discharge gas during the film deposition. The significant differences in wettability may be attributed to a high number of hydrophilic amine groups (including primary and secondary amine) on the PPAam-N2 surface. To verify this hypothesis, we further carried out amine quantification by AO II, which is a rapid and convenient C


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Figure 2. (A) Amine group quantitation by AO II and (B) GATR-FTIR spectra of the PPAam and the PPAam-N2 films.

vibrations of ν−NH at 3350 cm−1 indicates a good retention of the primary amine groups (−NH2). Comparing the PPAam and the PPAam-N2 films, a few differences in nitrogen containing groups can be observed. The peaks of ν−NH at 3350 cm−1, −N−H deformation vibration at 1625 cm−1, and −CN stretching vibration in 2280−2100 cm−1 were reinforced on the PPAam-N2 surface, indicating that the introduced nitrogen species contribute to the formation of amine, amide, imine or nitrile groups. Table 1 shows the C, N, O components of different samples and the corresponding nitrogen to carbon (N/C) ratios as well as allylamine monomer. As expected, the PPAam-N2 surface presented a nitrogen content of 15.8%, which was 8.2% higher than that of the PPAam and a N/C ratio of 21.2, which was 10.8% higher than for PPAam. To better determine the surface chemical composition of the PPAam and PPAam-N2 films, we carried out the analysis of the high-resolution spectra of N 1s and C 1s (Figure S2). The N 1s reveals at least three overlapping peaks (Figure S2A). One at 398.4 eV associated with C−N or CN groups, the main peak at 399 eV due to nitrogen atoms bonded to the carbon and the oxygen atoms (−CN, −N−CO, −N−CO−N−), and the broadest peak at 401 eV corresponding to O−C−O−N and CO−N−CO groups. Indeed, the increased content of −C−N, −CN groups, i.e., 45.7% (PPAam-N2) vs 39.8% (PPAam) contributes to the overall incorporation of nitrogen according to the analysis of fitting peaks of XPS (Table S2). Therefore, the role of nitrogen in the plasma-polymerized allylamine process has been confirmed to increase the possibility of nitrogen species that can react with

colorimetric assay based on the reversible electrostatic interactions.34 The values of the PPAam and PPAam-N2 films were 25.5 ± 2.8 and 37.8 ± 1.5 nmol/cm2, respectively (Figure 2A). The relationship between water contact angle and amine density of the series PPAam-N2 films is shown in Figure S1. All the PPAam-N2 series samples present a higher amine density than the PPAam surface and displayed higher hydrophilicity. Interestingly, we noticed that the PPAam-N2-5 sccm displayed the highest amine density. More nitrogen reactive species present a positive effect on deposition rate and at the same time, nitrogen containing groups especially amine groups were well retained and reproduced, when depositing plasma polymerized allylmaine films. GATR-FTIR (Figure 2B) and XPS (Table 1) were carried out to analyze the surface chemical Table 1. Elemental Compositions Obtained by XPS of the PPAAM and the PPAAM-N2 Surfacesa

a

sample

C (%)

N (%)

O (%)

N/C (%)

allylamine PPAam PPAam-N2

75 76.8 74.7

25 14.5 15.8

8.7 9.5

33.3 18.9 21.2

The allylamine group presents the theoretical values.

structures and compositions. As shown in Figure 2B, the basic features of allylamine (H2CCH−CH2−NH2) structure were well-reproduced in the two families of the PPAam films:35 (1) The peaks at 2925 cm−1 standing for C−H peaks was also the evidence that the radical chain growth polymerization occur during the “plasma-off” periods. (2) The presence of stretching

Figure 3. (A) Polarization curves and (B) Nyquist impedance spectrum of the bare 316L SS, the PPAam and the PPAam-N2 films in PBS at 37 °C (inset is impedance spectrum at high frequencies). D


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more absorbed on both of the plasma coating modified SS. To confirm the different absorbed masses between the PPAam and the PPAam-N2 films as well as the dynamic adsorption models, QCM-D was used to monitor the whole process (Figure 4B). With the same tendency measured by Micro-BCA arrays, the PPAam-N2 surface promoted the protein adsorption, by about 170 ng/cm2. 3.4. Cell Morphology, Adhesion, and Viability. To explore the potential application of the plasma coated surfaces on cardiovascular and hard-tissue implant and materials, we used the typical ECs and OBs to appraise the cytocompatibility.37,38 To evaluate the attachment, adhesion, and proliferation of ECs, we seeded ECs on the PPAam- and PPAam-N2-coated samples in comparison to the SS control for 2 h, 1 day, and 3 days, respectively. For the initial attachment stage of ECs (2 h), the amount of ECs attached on the plasmacoated surface was larger than that on SS, which may be attributed to the positive amine surfaces. Meanwhile, there was a higher EC count on the PPAam-N2 than on the PPAam surfaces (Figure 5A). CCK-8 test were used to evaluate the early growth stage (1 day) and the ability to proliferate (3 days). Here, the CCK-8 value at day 3 was normalized to the mean value at day 1 to calculate the proliferation rate of the adherent ECs. As shown in Figure 5B, C, ECs grown on the PPAam-N2 surfaces proliferated faster than those grown on the PPAam surfaces, whereas ECs on the SS surface proliferated slower than both of these two plasma-coating-modified samples. To further observe the morphology of cells on different samples, DAPI (2 h) and rhodamine123 (1 day and 3 days) were used to stain the cell nucleus and matrix. As shown in Figure 6, the PPAam-N2 sample showed more EC attachment than PPAam samples during the first 2 h incubation. The SS displayed the lowest adhesion amount of cells according to the fluorescent staining images. On the first and third culture day, ECs on all the surfaces presented an elliptical, cobblestone and polygonal morphology, indicating good cytoskeleton development. However, on the SS surface, a larger number of cells are apoptotic or necrotic, concluded from the bright spot morphology.39 This may be the main reason for low metabolic activity determined by CCK-8. In contrast, most of the ECs cultured on plasma-coating-modified samples exhibited increased cell spreading areas and better development of the cell cytoskeleton, suggesting a healthy EC growth on both PPAam and PPAam-N2 samples.40 These phenomena could be

other allylamine fragment and thus result in a substantial increase in nitrogen containing functional groups. 3.2. Electrochemical Corrosion Measurements. Representative potentiodynamic polarization curves of the PPAam and the PPAam-N2 deposited and unmodified SS are shown in Figure 3A. To evaluate the anticorrosion characteristics of these two families of the nitrogen-rich films coated on the SS, we have carried out the potentiodynamic polarization test. According to the Tafel extrapolation method, natural corrosion potentials (Ecorr) natural corrosion current (Icorr) were determined and the calculated values are listed in Table 2. Table 2. Polarization Tests Fitted Parameters for the Bare 316L SS, the PPAam, and the PPAam-N2 Films in PBS at 37°C params −2

sample

Icorr (μA cm )

Ecorr (V)

316L SS PPAam PPAam-N2

8.9 × 10−7 1.23 × 10−8 7.079 × 10−9

−0.37 −0.34 −0.138

The PPAam-N2 coated SS was over 2 orders of magnitude lower than that of the unmodified SS, and one order lower than that of the PPAam-deposited SS. From the thermodynamic point of view, Ecorr of the PPAam-N2 deposited SS was higher than that of the unmodified and PPAam-coated SS by 232 and 30 mV, respectively. EIS were also performed to obtain further information about the barrier properties and corrosion resistance performance of the plasma-polymerized films.36 The corresponding Nyquist plots are presented in Figure 3B. A significantly larger capacitive loop arc in the Nyquist mode confirmed the enhanced anticorrosion properties of the SS with the deposited PPAam and PPAam-N2 films. These results from the corrosion studies suggest an improved corrosion resistance of both the PPAam and the PPAam-N2 deposited SS. Besides, under the mixed vapor of allylamine and nitrogen, the deposited films of PPAam-N2 possess the better protective barrier properties in terms of corrosion resistance as compared to the simple PPAam samples. 3.3. Protein Adsorption. The protein adsorption results measured by Micro-BCA arrays for SS, the PPAam, and the PPAam-N2 films are shown in Figure 4A. Although the PPAam and the PPAam-N2 films tended to adsorb only little more BSA than the bare SS, proteins from complete serum were much

Figure 4. (A) Static protein adsorption of the SS, the PPAam, and the PPAam-N2 after 30 min incubation with model proteins (1% BSA and 5% FBS). (B) Dynamic adsorption of FBS on the PPAam and the PPAam-N2-modified Au surface via QCM-D real-time monitoring. E


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Figure 5. (A) Attached EC numbers on bare SS, PPAam and PPAam-N2 surfaces for 2 h. (B) EC proliferation on bare SS, PPAam, and PPAam-N2 surfaces at various periods of culture. (C) CCK-8 value at day 3 was normalized to day 1 to calculate the proliferation rate.

Figure 8. Cytoskeletal actin (green) and nuclear (blue) stains of OBs on the bare SS, PPAam, and PPAam-N2 surfaces after 1 and 3 days of culture.

Figure 6. Cytoskeletal actin (green) and nuclear (blue) stains of the ECs on the bare SS, PPAam, and PPAam-N2 surfaces after 2 h, 1 day, and 3 day culture.

3.5. Macrophage Response and Cytokine Release. Macrophage activities are closely related to immune and inflammation responses, which are remarkable and complex organization of defensive strategies.41 The immunofluorescence photographs and SEM images of macrophages after 24 h of in vitro incubation on the SS, PPAam, and PPAam-N2 surfaces are shown in Figure 9. Macrophages extended pseudopods and frontal cytoplasmic fringe or showed irregular shape on the SS sample, whereas they exhibited fewer pseudopods and had an extended or round shape on the PPAam and the PPAam-N2 surfaces, indicating the normal cellular shape of mainly resting cells.42 Also, the statistical results of immunofluorescence images revealed that the number of macrophages adherent on the SS was much larger than that on the PPAam and the PPAam-N2 surfaces. Both of the two plasma-polymerized films inhibited the adhesion and activation of macrophages, and no significant differences were observed between the PPAam and the PPAam-N2 samples.

attributed to the more hydrophilic surfaces and the higher density of amine groups on the plasma-polymerized allylamine surface. For the growth behavior evaluation of the OBs, there is no significant difference between SS and the different plasmacoated surfaces. The plasma-coated samples and the control SS strongly promoted OB growth. This could be well-reflected by the CCK-8 tests (Figure 7A) and the calculated proliferation rate (Figure 7B). Also, the fluorescent staining images of 3 days’ culture showed that the OBs grown on the plasma coating modified samples completely covered their surfaces, implying that the amine-rich surfaces provided a friendly microenvironment for OB growth (Figure 8). It may be the intrinsic more powerful proliferation ability of OBs compared to ECs, which decreased the effect of surface functional groups on modulating the cellular growth behavior. So no statistical differences of CCK-8 values were observed between SS, PPAam, and PPAamN2.

Figure 7. (A) Osteoblast proliferation on bare SS, PPAam, and PPAam-N2 surfaces at various cultural periods. (B) CCK-8 value at day 3 was normalized to day 1 to calculate the proliferation rate. F


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Figure 9. (A) Rhodamine123 and DAPI fluorescence staining of macrophage cultured on the bare SS, PPAam, and PPAam-N2 surfaces for 1 day culture. (B) SEM images of adhered macrophages on different samples. (C) Adhered cell numbers (left), TNF-α release (middle), and IL-6 release (right) of different samples after 1 day macrophage culture.

Cytokine released from macrophages to the cell culture medium were measured for further investigating the activation of macrophages. Figure 9C shows the TNF-α and IL-6 release from RAW 264.7 cells. Clearly, there was no significant difference in TNF-α release between SS and PPAam during 1 day culture. However, TNF-α secreted by the macrophages on the PPAam-N2 surfaces was lower as compared to the SS and the PPAam samples. Moreover, IL-6 was reduced for both plasma coated surfaces. The morphology of the adhered macrophages and the corresponding cytokine determinations confirmed that the plasma-polymerized allylamine surfaces exhibited the good noninflammatory properties. Probably, the anti-inflammatory effect of the plasma-polymerized films was related to the surface content of nitrogen. 3.6. In Vivo Animal Study. Bare SS and PPAam- or PPAam-N2-coated SS were implanted subcutaneously into the backs of rabbits to examine the interactions with the surrounding tissues. The samples were harvested from the subcutaneous pockets after 2 and 9 weeks of implantation for histological examination of contacting tissue using hematoxylin and eosin staining for the inflammation assessment and Masson’s trichrome staining for the analysis of fibrous encapsulation. Figure 10 shows the typical optical microscope images of the control SS, PPAam, and PPAam-N2 samples. In the case of the SS and PPAam groups, thick fibrocyte layers were observed at the implant−tissue interfaces, indicating severe acute inflammation 2 weeks after surgery. In contrast, only a thin fibrous capsule was formed on the PPAam-N2, which suggested a slight inflammatory response and no local toxic effects. When the implantation time was extended to 9 weeks, a thinning process of the fibrous capsule could be

Figure 10. Inflammation and fibrous propagation after 2 and 9 weeks of implantation of the SS, PPAam, and PPAam-N2 samples in the back of New Zealand white rabbits (the M regions refer to the implant).

observed in all the experimental groups due to the self-healing mechanism and the sufficient biocompatibility of the samples.43 The analysis of histological slides revealed that the SS and PPAam still presented obvious encapsulation, whereas the PPAam-N2 samples showed the weakest tissue responses with the thinnest fibrous capsule. Overall, the PPAam and PPAamN2 samples both displayed superior tissue responses compared to the control SS. G


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4. DISCUSSION Improvement of biocompatibility generally is a main intention for surface modification of biomedical implants.44 For the application as cardiovascular or hard-tissue implant, there is a main focus on the support of EC and OB adhesion and proliferation and decrease in inflammatory response.45−49 Most strategies are based on immobilization of growth factors (such as vascular endothelial growth factor, bone morphogenetic protein, transforming growth factor, and fibroblast growth factor)50−53 or the incorporation of antibodies, antibiotics, or functional element.54 Few modifying coatings that can load functional molecules or link bioactive molecules play multifunctional roles and provide synergistic effects. Plasma polymerization techniques have been successfully adopted in modifying physical and chemical properties of the surfaces of implants to achieve this goal. In this work, we highlight the plasma-polymerized, nitrogen-rich films which present good cellular adhesive properties and in addition attenuate inflammatory and histological responses. This one-step dry process and substrate-independent modification method also provides anticorrosion properties to the substrate. Moreover, as a platform, this type of plasma-polymerized coating contains abundant amine functional groups for the further conjugation of functional molecules. These potential advantages can allow better biofunctionalization of implants behaving in different complex microenvironment in vivo. Previously, many attempts have been made to prepare a nitrogen-rich plasma polymer by adjusting glow discharge parameters and introducing nitrogen species.15 In optimizing our amine-rich films, we introduce nitrogen as reactive gas and a large amount of nitrogen species act as reactive centers followed by bonding of fragment of the allylamine monomer. The increase in the film deposition rate confirms the supportive effect of nitrogen in the reactive gas mixture. The higher nitrogen content in XPS and the formation of complex C/N/O bindings confirms the integration of nitrogen into the polymer and its support of the polymerization process. The high amine content in the AO II assay indicates the accessibility of the groups also at the surface. This indicates that nitrogen, as an assistant deposited gas, plays a critical role in the growth of the plasma-polymerized allylamine films and in the expression of reactive amine groups. The PPAam-N 2 showed better protective barrier properties compared with the PPAam and SS based on the electrochemical corrosion assay, which may be attributed to the more densely cross-linked network of the films by aid of nitrogen cross-linking during plasma deposition process. In recent years, plasma polymerized coatings using monomer of siloxane, fluorocarbon, and others have been widely reported to improve corrosion resistance.55,56 The corrosion protective properties were only evaluated on substrates of magnesium and nickel titanium.57,58 In this study, the PPAam has been demonstrated to be used as an anticorrosion film also on SS medical devices. Also the PPAamN2 modification strategy exhibited the strong corrosion protection, and hence may inhibit the toxic nickel ions release from 316L SS surface and may decrease the nickel-associated complications like allergies or in-stent restenosis of bare metal stents. Biomaterial surface engineering has been used to endow the implants with specific functionalities through the changes in surface properties while maintaining the bulk properties.59 Actually, the outermost layer of the material mainly governs the

interaction with biological tissues or biological responses to the synthetic biomaterials and the living system. Amine groups are generally considered to play a critical role in regulating the growth behavior of cells. They are also one of the most important groups for the conjugation of bioactive molecules to the surface.60 Many research groups have successfully prepared plasma-polymerized allylamine films to enhance biocompatibility and promote growth of cells.61,62 Our films present similar results of promoting EC and OB attachment, which may be a consequence of high density of amine groups. Recent research about the effect of surface function groups on EC growth and migration is lucubrated at the molecular level. The results of self-assembled monolayers show that ECs migration is enhanced in the order CH3 > NH2 > OH > COOH, according to the expression of focal adhesion components, Rho GTPases and (p)FAK.63 The adsorption behavior of proteins is considered to mediate the cell adhesion and growth on biomaterials.64 For further explaining the phenomena of promoting cell adhesion and growth on the plasmapolymerized surface, we detected the absorbed amount of single component albumin solution since albumin is one of the most abundant proteins in plasma. The results demonstrated that the more amine groups on the surface of the plasma polymerized films, the more proteins tend to bind and the similar tendency was found when incubate cell culture medium that consist of multiple protein components. Dynamic monitoring of protein adsorption on these two families of plasma coating rich in amine groups by QCM-D also confirmed that the high amine density on the surface caused larger amount of protein adsorption. At subcellular level, we found that this protein affinity of the surfaces correlates well with cellular behavior. Therefore, we may conclude that the more adsorbed proteins on plasma polymerized allylamine films contribute to regulating cellular behavior including boosting cells responses in their initial adhesion phase and representing biocompatible surface that promotes rapid attachment of ECs and OBs. These results are in an agreement with our previous research about the modulation of protein adsorption and vascular cell by mussel-inspired polydopamine surface.65 Binding of more serum proteins on the polydopamine surfaces triggered favorable vascular cell attachment, focal adhesion development, stress fiber formation, and cell growth. In the application of plasma polymer-coated metals as cardiovascular or hard-tissue implant, surfaces are frequently chemically and physically modified with biomolecules and ligands for cell adhesion receptor. This subsequently provides biomodulating or biomimetic microenvironments to contacting cells and tissues.66 Clinical implementation for cardiovascular regeneration requires materials with structure and function similar to native vascular tissue. The noncytotoxic responses to ECs and support for establishing a monolayer of ECs are basic demands as a platform for the cardiovascular devices. However, the overproliferation of smooth muscle cells may lead to the low patency and stenosis of implants like vascular stents or grafts.67 In an effort to expedite endothelialization and enhance the properties of EC competitiveness over that of SMC, further modification is essential to endow the device with a multifunction. In our recent works we have presented many approaches to immobilize bioactive molecules onto the PPAam surface, such as heparin, gallic acid, bivalirudin, and nitric oxide generating systems to construct both hemocompatible and ECs promoted surfaces.25,30,35,68 A 48.2% increase of amine groups in the PPAam-N2 surface allow a relevant amount of these H


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its better anti-inflammatory properties.73,74 Characterization of these effects at the molecular level is intended for further work. In the present study, we demonstrated the relevance of the antiinflmmatory behavior by the in vivo animal tests. Surgical implantation of biomedical devices causes cell and tissue injury and thus triggers the wound healing response of surrounding vascularized tissue. Hoene et al.75 evaluated the effect of a plasma-polymerized allylamine layer on the local inflammation in a rat model. They have demonstrated that variations in the material properties such as water adsorption, nitrogen loss, and oxygen uptake caused by the plasma conditions have a great influence on the host tissue reactions. Afterward, their group prepared plasma-polymerized ethylenediamine (PPEDA) coatings and investigated the inflammation reactions by the same method.76 Their results indicated that the increased cell adhesion as observed in vitro for the PPEDA surface leads to short- and long-term adverse effects in vivo concerning the attraction of inflammatory cells, which were not found for their previous PPAam surfaces. This was attributed to a higher amine density of PPEDA compared to PPAam because of the different chemistry of the respective precursor molecules. In our in vivo animal tests, we therefore focused more on the different inflammatory reactions resulting from surfaces with different amine density. Figures 9 and 10 show that the surfaces with amine groups (PPAam and PPAam-N2) may decrease effects of complement activation and lead to weaker macrophages activation, lower release of cytokines, and better tissue compatibility than SS. A similar result regarding attenuated inflammatory reactions was observed between our PPAam and PPAam-N2 samples and their PPAam samples. Increased amine density did not result in more serious inflammation, but in turn could cause less pronounced inflammatory reactions in both acute and chronic phases according to our in vivo results. In our opinion, residual monomers or minor difference of surface characteristics may lead to the different observations. The adhesion and activation of macrophages and cytokine release are thought to affect the foreign body response to implanted medical devices. However, these cells interact with devices indirectly, because of the initial adsorption of proteins.77 As it was shown that more proteins adsorbed onto the surfaces with high amine density, probably the signal of foreign body reaction was shielded and therefore the inflammatory response was decreased. In 2008, Nair et al. investigated the influence of density of surface functional groups (−OH, −COOH and −CH 3 ) on host tissue responses.78 They found that the type, but not the density, of the functional groups has a significant effect on the inflammatory cell recruitment and tissue reactions. For the surfaces with different amine densities in the present study, the foreign body reactions strongly depended on the amine density. The high amine density increases the surface free energy (polar components) and renders the surface more hydrophilic. Generally, hydrophobic materials enhance the monocyte adhesion at the implant site, while hydrophilic surfaces have decreased macrophage adhesion and show reduced foreign body giant cell formation in vitro.79 Furthermore, the BSA adsorption is directly related to the surface energy, with an excellent linear correlation.80 This kind of protein seems to passivate the surface and inhibit the macrophage adhesion and the relative cytokines release.77 That may be the reason that PPAam-N2 samples exhibited better anti-inflammation properties than PPAam samples. The surface with higher density of amine groups could absorb more BSA and thus perhaps limit

molecules to be immobilized using amine reactive carbodiimide chemistry. Further research about the biomolecule immobilization and their effects on the improvement of biocompatibility will be carried out systematically. In the case of new bone formation and hard-tissue engineering, the crucial factor of metal implant ingrowth in the bone is the rapid osteocytes cellular responses.22 It has been demonstrated that PPAammodified titanium supports osteoblastic focal adhesion formation as vinculin and paxillin, actin cytoskeleton development, and in consequence, in specific cell functions (bone sialo protein expression). This depends on the interactions between the negatively charged hyaluronan coating of human MG-63 OB and positively charged PPAam surface in culture media. The more amine-rich PPAam-N2 surface provides a higher density of positive charges and thus is much easier protonated into high positively charged surface, which strengthens the interactions between modified surface and OBs. However, osseointegration involves both osteoconduction and osteoinduction.69 OB migration and sprout from the surrounding tissues onto the implants play a vital role in osteoconduction. Besides OBs adhesion and proliferation, osteogenic differentiation of relative stem cells, especially bone-marrow mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs), into osteoblastic lineage could be helpful in osteoinduction, which is necessary for large critical-size bone repair.62,23 At present, tailoring of osteogenic differentiation of stem cells on nitrogen-rich films via plasma polymerization technique has been paid much attention. A positively charged surface derived from nitrogen plasma treatment on a polymeric orthopedic implant shows the immense potential as a local biochemical and electrical environment to activate iNOS expression and signal BMSCs to differentiate via the osteogenic pathway, thus creating a positively charged surface with amines surfaces is a promising approach to promote osseointegration with bone tissues.70 Considering the multiple functions of this platform, our research of these plasma films rich in nitrogen could form a basis for products that are the promising candidates for clinical application with improved endothelialization in the case of vascular implants or enhanced new bone tissue formation in the case of hard tissue implants. The clinical success of hard tissue implants is easily abolished by an inflammatory response of the local environment, as it influences the long-term stability and functionality of the implant. The presence and function of macrophages at the implantation site during wound healing constitutes the main part of what is referred to as foreign body reaction. In earlier reports, the serum profile of pro- and anti-inflammatory cytokines in rats following implantation of plasma-allylamineor acrylic-acid-modified titanium was systematically examined.71 PPAam-modified devices were implanted into the Sprague− Dawley rats for evaluating inflammatory response and bone-toimplant contact as well.72 In the present study, we also evaluated the interactions of the plasma-polymerized nitrogen surface with macrophages and demonstrated that the plasmacoated surface had an anti-inflammatory effect on RAW 264.7 cells. To examine such effects, we assessed the levels of wellknown pro-inflammatory cytokines such as TNF-α and IL-6. The results showed that less cytokines expression was detected on PPAam and PPAam-N2 samples. In contrast, slight upregulation of IL-6 was detected on macrophages cultured on the SS, possibly mediated by material-dependent interactions with certain immune cells. The lower release of TNF-α on the PPAam-N2 sample compared to the PPAam also demonstrates I


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ACS Biomaterials Science & Engineering the inflammations. The surface chemistry characteristics determines the early responses to the implantation and following healing process (specifically: protein deposition, coagulation, interactions of inflammatory and tissue cells with the material surface), but many late aspects of wound healing, specifically, foreign body reaction and fibrous encapsulation, are influenced by the rate, accumulation, and bioactivity of released chemicals and corrosion products from prosthetic devices from the implant.81 Metal ions such as nickel, chromium, and cobalt, which are constituents of 316L SS, can induce hypersensitivity responses and are seen as being responsible for chronic inflammation post-implantation.82 In our in vitro corrosion tests, we found that the plasma polymerized nitrogen-allylamine films remarkably reduce the corrosion of SS, which may be another reason for its good tissue-compatibility. The existence of the protective films of plasma polymerized films may prevent the release of toxic ions and reduce the local inflammatory reactions surrounding the implant site. Therefore, the superior biocompatibility of PPAam-N2 sample may be attributed to the high amine density in the surface combined with the corrosion protective property of the polymerized films. However, several limitations of the present study should be considered. We have not yet identified whether general compositional alterations of the PPAam layer occur during a storage time or a sterilization by ethylene epoxide process.83 Second, it is not possible from the data of both studies to establish a definitive link between the cellular response on that day and corresponding changes in the implant surface properties. Also this study is limited to observations in experimental animal models and inadequately big sample data, thus its clinical relevance to human beings is uncertain. For the animal tests, further investigations are necessary to systematically evaluate the tissue-compatibility of the simple amine surfaces and also the physiological stability of plasma-polymerized nitrogen films. The changes of surface characteristics of plasma-polymerized nitrogen-allylamine films during the interactions with tissues as well as the toxicity of released metal ions to the surrounding microenvironment should be carried out during the following research.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: +86 28 87600625. Fax: +86 28 87600625. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Manfred. F. Maitz for his help in providing language corrections and valuable discussions. This work was supported by the National Natural Science Foundation of China (Project 81271701, 51173149, and 31470921), the Ministry of Science and Technology of China (Key Basic Research Project 2011CB606204), and NSFC Key Program 81330031.

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REFERENCES

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5. CONCLUSIONS Plasma polymerization of allylamine directly or in the presence of nitrogen in the plasma can be used for the formation of films containing various amounts of amine groups on a wide range of biomedical devices. The coating provides corrosion resistance to the surface and shows good biocompatibility as it promotes EC and OB attachment and proliferation and helps the devices to behave well in the local environment. These findings suggest that plasma polymerization technology is a viable approach to improve both the tissue response and cytocompatibility. The excellent performance endowed by this approach can be used in the surface modification of biomedical devices ranging from cardiovascular materials to hard-tissue implants and extended across various applications in sophisticated biosensors and tissue engineering fields.

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quantification, and high-resolution XPS N 1s and C 1s peaks (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ab5001595. Different deposition parameters of plasma-polymerized allylamine-nitrogen films, water contact angle, amine J


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