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O2 glow discharge plasma
Applied Surface Science 255 (2009) 7257–7262
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Biological response of stainless steel surface modified by N2O/O2 glow discharge plasma Meng Chen *, Shigemasa (Gem) Osaki, Paul O. Zamora BioSurface Engineering Technologies (BioSET), Inc., 9430 Key West Avenue, Rockville, MD 20850, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2008 Received in revised form 18 March 2009 Accepted 24 March 2009 Available online 1 April 2009
Stainless steel wafers were treated with the glow discharge plasma of mixed N2O and O2 at different molar ratios at a certain discharge condition to create desirable biological characteristics to the surfaces. It was found that the molar ratio of N2O to O2 in the mixture at 1:1 used for plasma surface modification caused high apoptotic percentage. Contact angle measurement showed that the surface of stainless steel samples became very hydrophilic after the plasma modification with a value of 158–308. The control stainless steel chips without plasma treatment had a contact angle of 40 28. The data of Electron Spectroscopy for Chemical Analysis (ESCA) indicated that there was a certain amount of oxynitrites formed on the plasma treated surfaces, which was considered to play an important role to cell apoptosis and anti-clot formation in cell culture tests. The ESCA depth profile of up to 250 A˚ from the top surface showed the change of elemental compositions within 40–50 A˚ of the surface by the plasma treatment. The decreased platelet attachment, combined with increased apoptosis in fibroblasts is a distinct combination of biological responses arising from the mixed gas plasma treatment. These initial results suggest it may be of particular use relative to stainless steel stents where decreased platelet attachments are advantageous and induction of apoptosis could limit in-stent restenosis. ß 2009 Elsevier B.V. All rights reserved.
PACS: 52.40.Hf 33.60 Fy 87.14. g Keywords: Surface modification ESCA Nitrous oxide Cell culture Apoptosis Glow discharge plasma
1. Introduction Plasma treatment is a unique technique used to alter biomaterial surface property and its consequent biological response. Glow discharge plasma, known as low temperature plasma, is generated by energizing a gas inside a vacuum chamber. The active components of plasma include ions, electrons, radicals, excited species and photons, among others. The collective properties of these active species can be controlled and harnessed so as to perform a variety of surface treatments, including nanoscale cleaning, activation for surface wettability, chemical grafting, and creating surface bioactivity. Because of the highly reactive environment, plasma can be employed to change the properties of surfaces without affecting the bulk materials. Furthermore, plasma is a dry surface treatment technique, so there are no waste chemicals to dispose of, making this an environmentally friendly process involving very few consumables. Owing to these advantages the plasma surface modification process has been widely used in recent decades for the preparation of biomedical materials
* Corresponding author at: ExcelCoat, LLC, 30670 Ardmore Ct., Novi, MI 48377, United States. E-mail address: [email protected] (M. Chen). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.03.079
with unique surface performances and in the manufacturing of medical devices [1,2]. Oxygen plasmas were used to increase the attachment of cells to polymer surfaces [3–5]. Plasmas were also utilized to introduce amines and amides to polymeric materials for increasing the attachment of cells, and in particular endothelial cells [6–10]. It has also been reported that the plasma treatment resulted in a change in the amount and type of blood proteins that are adsorbed on the treated biomaterial surfaces. For instance, fibronectin and vitronectin are two such blood proteins whose absorption is modified by glow discharge [11,12] thus influencing endothelial cell attachment. Oxygen plasma treatment of polymers was also found conducive to the binding of laminin, another protein that influences cell attachment [11]. Surface properties of poly(D,L-lactide) modified by combining plasma treatment and collagen modification demonstrated considerable improvement in cell affinity [13]. Degradable poly(hydroxybutyrate) thin film treated with low pressure ammonia plasma or low pressure water vapor plasma showed increased hydrophilicity and improved cell adhesion [14]. Another biodegradable polymer, poly(epsiloncaprolactone) (PCL) was modified by an O2 plasma surface treatment to form oxygen-containing functional groups. Consequently, the apatite layer strongly adhered to the PCL surface in the resulting apatite-PCL composite [15]. The surface modification of medical-grade poly(vinyl chloride) (PVC) using a radio-frequency
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oxygen glow discharge pre-functionalization followed by a twostep wet-treatment in sodium hydroxide and silver nitrate solutions was proved to prevent bacterial adhesion to PVC tubes [16]. The surface modification of biocompatible PCL film treated by atmospheric cold plasma with reactive gases was also reported to improve cell distribution and growth [17]. In addition to synthetic polymers, metals like stainless steel and titanium or alloys widely used in the construction of medical devices [18] are also treated with plasmas for a variety of purposes. Plasma treatment of stainless steel and titanium resulted in surfaces with enhanced resistance to platelet and leukocyte attachment [19]. Surface modification of stainless steel by argon plasma pretreatment followed by grafting of poly(ethylene glycol) was found to be very effective in preventing protein adsorption [20]. Surface cleaning and modification of metallic biomaterials using glow discharge plasma treatment provide a clean and reproducible starting condition for further plasma treatments to form well-controlled surface layers [21]. Polished titanium samples were treated using a plasma exposure apparatus in gas atmospheres of air, CO2, and C3F8. It was observed that plasma exposure in a specific gas atmosphere regulated the bonding strength of titanium surface to resin in dental applications [22]. Simvastatin acid (SVA) has been reported to stimulate bone formation. The largest amount of SVA was adsorbed on O2 plasma treated hexamethyldisiloxane (HMDSO) surfaces compared to untreated titanium, HMDSOcoated titanium, and O2 plasma treated titanium [23]. Plasmacoated nitinol, alloy of nickel and titanium, intravascular stents showed increased surface hydrophilicity and enhanced anticoagulation property [24]. The objective of this study is to impart unique biological characteristics to the surface of stainless steel material, which includes decreased platelet attachment and increased apoptosis in fibroblasts. Plasmas of nitrous oxide mixed with oxygen (N2O/O2) at various molar ratios were used to modify the surface of stainless steel chips. The effect of the plasma surface treatment was characterized by means of surface contact angles and Electron Spectroscopy for Chemical Analysis (ESCA). The biological responses of stainless steel before and after the surface modification were evaluated by means of clot formation test and cell apoptosis test of C3H10T1/2 fibroblasts. The correlation between surface functional groups formed from plasma treatment and cell apoptotic event was discussed. 2. Experimental 2.1. Materials
Table 1 Material analysis of 316 stainless steel used in this study. Element
Atomic fraction
Carbon (C) Manganese (Mn) Phosphorus (P) Sulfur (S) Silicon (Si) Chromium (Cr) Nickel (Ni) Nitrogen (N) Copper (Cu) Iron (Fe)
0.08% max. 2.00% max. 0.045% max. 0.030% max. 1.00% max. 16.00–18.00% 10.00–14.00% 0.10% max. 2.00–3.00% Balance
USA), which was 10% of polyethylene glycol based detergent (CAS No.: 9003-11-6) in ethanol, and rinsed exhaustively with water, and air-dried. 2.3. Experimental setup and process parameters The plasma reactor used in this study is shown in Fig. 1. The radio-frequency (13.56 MHz) power supply was applied to plasma chamber through a matching network (not shown in the figure). Between the two high-voltage driven electrodes a discharge zone of 19 cm 16 cm 23 cm (width length height) was established. Prior to feeding of working gases to the plasma chamber, a base vacuum of better than 10 mTorr was achieved in about 5 min using vacuum pumps. The vacuum pumps include one turbomolecular pump (Osaka Vacuum, Model TS433) purchased from Osaka Vacuum, Ltd., San Jose, CA, USA, and one mechanical pump system consisting one rotary vane vacuum pump (Alcatel 2033CP+) and one roots blower (Alcatel RSV 300B) procured from Alcatel Vacuum Products, Fremont, CA, USA. A precision motorized throttle valve was used to regulate the plasma system pressure. The valve was electronically connected to a throttle valve controller. This controller was electronically connected to a main pressure transducer and to a control interface unit which was hooked up with a computer for controlling the operation of the entire plasma system. The computer read the system pressure from the main pressure transducer and compared with the pressure set point sent by the computer. The system feedback loop regulated the opening of throttle valve to maintain the system pressure at the set point pressure constantly. The working gases such as N2O, O2 or NH3 were introduced into the plasma chamber through the inlet located in the front of the chamber. The molar ratios or flow rate ratios of N2O over O2 used in the experiments varied from 0:1, 1:4, 1:2, 2:3, 1:1, 3:2, 4:1 to 1:0. The operational
Square wafers in the dimension of 7.1 mm 7.1 mm 2 mm were used in the studies. They are made of 316 stainless steel (SS), a ‘‘surgical stainless steel’’. This type of stainless steel will not rust or tarnish, is not magnetic, and is generally recognized as nonallergenic. Its elemental composition obtained from the Salem Specialty Ball Company, Canton, CT, is listed in Table 1. The stainless steel sheet, from which the square wafers were cut, was purchased from McMaster-Carr, a supply company at New Brunswick, NJ, USA. Nitrous oxide (N2O) with purity of 99.9995%, VLSI grade, and oxygen (O2) with purity of 99.9%, industry grade, were purchased from Air Products and Chemicals, Inc., Allentown, PA, USA. Ammonia (NH3) with concentration of 99.999%, electronic grade, was also supplied by Air Products and Chemicals, Inc. 2.2. Cleaning process for SS wafers All the SS wafers were cleaned with diluted (2% in distilled water) Acationox detergent (Baxter Healthcare Corp., Deerfield, IL,
Fig. 1. Schematic diagram of plasma reactor used in this study.
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plasma parameters were discharge power of 110 W, working pressure of 50 mTorr, and a total mass flow rate of 50 standard cubic centimeters per minute (sccm) for the gas mixture of N2O/O2 or NH3/O2. In all cases, the plasma treatment time was set at 45 s chosen on the optimal effect of plasma processing on stainless steel surface in regard to N or O elemental composition formed on the surface.
mixing platform in a lateral position (100 cycles/min) for 15 min then transferred to a 37 8C incubator. The mixing and warming were repeated for a total of 1 h. The wafers were rinsed 4 times in buffered saline and fixed in buffered formalin. Images of the wafers were then scanned as digitized images.
2.4. Contact angle measurement
3.1. Contact angle of plasma treated coupons
The contact angle measurement was performed using NRL 10000 Contact Angle Goniometer manufactured by Rame´-hart, Inc., Mountain Lakes, NJ, USA. The HPLC grade distilled water was used for the drops on sample surfaces. All the measurements were carried out at room temperature and the static water contact angle was obtained. Contact angle data were utilized to see how the stainless steel surface was affected by plasma treatment from the perspective of surface hydrophilicity and what correlation it might have to its surface bioactivity.
In Fig. 2, the contact angle data showed that the surface of stainless steel samples became very hydrophilic after plasma modification of N2O/O2 plasma with the average values for n = 5 from 158 to 308. In contrast, the control stainless steel chips without plasma treatment rendered a contact angle of 40 28. The measurement was carried out at 1 week after plasma treatment. Those plasma treated SS wafers were stored in plastic vials until use. The large standard deviations in the contact angle data observed for most conditions could be due to the unstable surface functionality created by NO2/O2 plasma treatment under those conditions. Contact angle analysis is widely used to evaluate the wettability of surface and has been suggested to be a key determinant of cell attachment [25,26]. Wettability is known to affect the adsorption of blood proteins thus regulating a variety of cell behaviors such as cell attachment. The experimental results from Webb et al. [25,26] indicated that contact angles from 20 to 40 rendered the highest anchorage-dependant-cell attachment for the materials they evaluated. In the present study, a highly hydrophilic surface was created on stainless steel by plasma treatment, but a much less blood clot formation, or a reduced level of platelet attachment, was observed on the plasma treated surfaces as compared to untreated controls (see Section 3.4 for blood clot result). This observation implies that the bioactive groups containing oxygen and/or nitrogen formed on the substrate surface other than surface hydrophilicity may play a critical role in platelet attachment and cell apoptosis.
2.5. Surface chemical composition studies ESCA was accessed under contract with the Department of Chemistry, University of Utah, Salt Lake City, UT, USA, in which the ESCA instrumentation was VG Scientific 220i-XL imaging multitechnique surface analysis system, using an Al Ka X-ray source. Material Interface, Inc., Sussex, WI, USA, was another source accessible to this study for performing ESCA measurement. The relevant equipment was Physical Electronics Model 5802 Multitechnique System with a monochromatic aluminum anode and an analysis area of approximately 0.8 mm 2 mm (width length), taking-off angle of 458 with respect to the incident angle of X-ray source. 2.6. Apoptosis test Apoptosis is the carefully regulated process of cell death. The appropriate regulation of apoptosis is important for the prevention of many disease states. In the present study, we use cell apoptotic percent as a criterion to evaluate the efficacy of plasma surface treatment on slowing down the healing process of vessel injuries resulting from implant placement in clinical applications. Plasma treated stainless steel wafers were disinfected, placed in wells of 24-well of low-attachment tissue-culture plates (Corning Inc., Corning, NY, USA), and aliquots of mouse fibroblasts (C3H10T1/2; ATCC, Manassas, VA, USA) added. At selected times the specimens were rinsed, fixed, stained with bis-benzimide (a DNA-binding fluorochrome), and viewed with a fluorescence microscope. Cells were scored as apoptotic if the nuclei were blistered, evidenced condensation of chromatin, and/or evidenced fragmentation of the nucleus.
3. Results and discussion
3.2. ESCA result The survey scan measurement results of ESCA are tabulated in Table 2. The data showed that the elemental composition of both nitrogen (N) and oxygen (O) was increased at the stainless steel surface after N2O/O2 plasma treatment, indicative of oxynitrites functional groups formed on the surface, which was considered to cause the cell apoptosis and decreased platelet adhesion and clot formation. Oxynitrites have been found to promote cellular damage and ultimately cell death by other researchers [27–30]. The elevated O and N component levels on the surface could also be the cause for decreased contact angle. As one derivative of
2.7. Clot formation test In this test, human blood was obtained by venipuncture and collected into specially prepared glass tubes containing a suboptimal amount of heparin (approximately 1/10 normal). This was designed to slow but not to stop clotting. The blood was collected into plastic centrifuge tubes to minimize (but not eliminate) platelet activation. Aliquots of 1 ml of blood were added to 15 ml capacity centrifuge tubes containing stainless steel wafers with or without plasma treatment. The plasma treatment was performed using gas mixture of N2O/O2 (12.5:12.5) for 45 s. To each tube had been added 4 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA, USA). The capped tubes were mixed on a
Fig. 2. Contact angle of control and plasma treated SS chips versus different ratios of N2O to O2. The data are presented as means standard deviations for n = 5.
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Table 2 Surface composition as determined by Electron Spectroscopy for Chemical Analysis (ESCA) of stainless steel wafers: control and N2O/O2 plasma treated (at molar ratio of 1:1). Sample identification
None N2O/O2
Atomic fraction for elements with 50 A˚ of the SS surface C
N
O
F
Si
S
Cl
Cr
Fe
Ni
Mo
21.9 17.0
0.5 1.3
52.5 57.9
0.6 0.4
1.9 3.5
1.0 0.1
0.2 0.0
10.7 7.2
9.5 10.4
1.1 1.9
0.2 0.3
oxynitrites, nitric oxide (NO) on the surface is known to have potent anti-platelet aggregating activity [31–37]. Nitric oxide (NO) release polymers to prevent thrombus formation have been found to cause a dramatic decrease in platelet adhesion and surface clot formation (compared to blank films) during both in vitro and in vivo experiments [37]. It is not clear how the carbon content at the substrate surface affects its bioactivity. To further study the potential influence of carbon at surface on cell apoptosis or other biological responses, a hydrogen plasma treatment will be applied to stainless steel substrates in follow-on studies since it could provide a more pristine surface with reduced carbon content. The trace amount of fluorine detected at the surface of both untreated and N2O/O2 plasma treated specimens could be attributed to the background noise from ESCA measurement. Depth profiling of the main elements including C, N, O, Si, Cr, Fe, and Ni for both untreated and treated SS wafers was plotted in Fig. 3. The SS samples were depth-profiled using ESCA with an argon ion beam at 20 A˚/min versus SiO2. Notably, the atomic fractions of C, N and Si for both plasma treated and untreated SS were around 1–2% in the near surface layer and did not exhibit difference between treated and untreated ones due to their low values. A deeper iron depleted surface layer (100 A˚) was observed for the N2O/O2 plasma treated SS sample than that (60 A˚) of the untreated control. In the case of plasma treated sample, the oxygen depth profile was broader than that for untreated control. The depth profile of the Cr and Ni of
plasma treated sample was shifted toward the inside too, which could be a result of oxygen incorporation in the near surface layer during the surface modification process using N2O/O2 plasma. These changes observed in the elemental components revealed that the N2O/O2 plasma surface treatment could modify the stainless steel surface to a depth of about 40–50 A˚ from the top surface generating a thin layer of oxygen-containing functional groups, such as oxynitrites, other than metal oxides. 3.3. Apoptosis The apoptotic percent of cells is plotted in Fig. 4 versus the molar ratio of N2O/O2. The untreated wafer is included as control, and the result of doxorubicin used as a positive control is also presented. It is found that the ratio of 1:1 (actual mass flow rate of N2O to O2 is 12.5 to 12.5 sccm) of N2O to O2 in the mixture resulted in the highest apoptotic percent in the cell culture experiments. Please note, the original data of the apoptotic percentage indicated that for each condition with two determinations the variation was less than or equal to 1%. Therefore, it is reasonable to believe that more than 1% apoptotic percentage difference from sample to sample would be significant. Fig. 4 also indicated that ammonia/ oxygen (NH3/O2) plasma treated stainless steel surface exhibited a similar increase in apoptosis to N2O/O2 (at the ratio of 1:1) plasma modified surface suggesting that this property may be common to
Fig. 3. Comparison of depth profiles for stainless steel chips with or without N2O/O2 Plasma treatment. Samples were depth-profiled using ESCA with an argon ion beam at 20 A˚/min versus SiO2.
M. Chen et al. / Applied Surface Science 255 (2009) 7257–7262
Fig. 4. Apoptosis in C3H10T1/2 fibroblasts at 1 day after seeding on stainless steel wafers with and without plasma treatment. Notes: control—no treatment, am-ox— ammonia/oxygen plasma, and doxorbn stands for doxorubicin which is used as a positive control, (1) N2O (0)/O2 (25), (2) N2O (5)/O2 (20), (3) N2O (8)/O2 (16), (4) N2O (10)/O2 (15), (5) N2O (12.5)/O2 (12.5), (6) N2O (15)/O2 (10), (7) N2O (20)/O2 (5), and (8) N2O (25)/O2 (0). The data are the average of two determinations with both of the values closely agreeing.
surfaces coated with oxynitrites. Actually it was found that oxynitrites were formed on NH3/O2 plasma treated stainless steel surfaces based on the ESCA data that had been reported elsewhere [19]. We also performed ESCA analysis on all the stainless steel samples treated with plasmas at different molar ratios of N2O to O2 as presented in Fig. 4. However, no clear correlation could be established between the elemental compositions of N and O and the corresponding apoptosis data for all the molar ratios used. As a result, we focused on the unique condition of N2O to O2 at the ratio of 1:1 in an effort to better study and understand the surface chemistry created by this plasma treatment and the plausible mechanism of its generating high apoptosis on the treated surface. To treat the major health problem in the United States, that is coronary heart disease, stenting has been widely accepted to reduce restenosis rate after percutaneous balloon angioplasty. The coronary artery stent, a small mesh tube made of stainless steel or other metal or alloys, functions as a scaffold to prop open blocked heart arteries to keep the passages from reclosing. However, the restenosis remains as an issue with stenting to some patients since the biological response to the vessel damage caused by stent implantation is stimulation of accelerated growth of arterial smooth muscle cells. Creating smooth muscle cell apoptosis to an adequate level on the surface of stainless steel is considered as an effective approach to inhibit cell proliferation and prevent rapid healing of tissue around the stents made of stainless steel. 3.4. Clot formation Fig. 5 is the image of stainless steel wafers after blood clot test. Apparently, the wafers without plasma treatment exhibited blood clot, whereas the treatment of stainless steel with nitrous oxide/ oxygen (N2O/O2) plasma resulted in resistance to clot formation in vitro. This result indicated that the level of platelet attachment to stainless steel following treatment with the N2O/O2 plasma was reduced considerably as compared to untreated controls. Also shown in Fig. 5 is a reduced clot formation on the SS wafers treated with plasma of NH3 and O2, suggesting that other combinations of nitrogen-containing gases plus oxygen or oxygen-containing gases could mimic the reduced clot formation due to the formation of similar functional groups such as bioactive oxynitrites on the substrate surface. The metal atoms on the substrate were found not critical in decreasing platelet attachment, since stainless steel
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Fig. 5. Treatment of stainless steel with either ammonia/oxygen plasma or nitrous oxide/oxygen plasma resulted in resistance to clot formation in vitro.
wafers coated with siloxane plasma coatings to mask the metal atoms also demonstrated reduced levels of platelet attachment on these materials [19]. 4. Conclusions Stainless steel surfaces were modified with glow discharge plasma of nitrous oxide mixed with oxygen at different molar ratios. The mixed gas plasmas of N2O/O2 with optimal concentration imparted unique characteristics to stainless steel by generating bioactive functional groups containing oxygen and nitrogen such as oxynitrites in the near surface layer. The non-clot formation on plasma treated surface, combined with increased apoptosis in fibroblasts is a distinct combination of biological responses arising from the mixed gas plasma treatment. These initial results suggest N2O/O2 plasma may be of particular use relative to stainless steel stents where the induction of apoptosis could limit in-stent restenosis thus improving the long-term safety in its clinical applications. References [1] B.D. Ratner, in: R. d’Agostino, P. Favia, F. Fracassi (Eds.), Plasma Processing of Polymers, Kluwer Academic Publishers, Dordrecht, 1997. [2] P.K. Chu, J.Y. Chen, L.P. Wang, N. Huang, Mater. Sci. Eng. R36 (2002) 143–206. [3] S.I. Ertel, A. Chilkoti, T.A. Horbett, B.D. Ratner, J. Biomater. Sci. Polym. Ed. 3 (1991) 163–183. [4] A. Chilkoti, A.E. Schmierer, V.H. Perez-Luna, B.D. Ratner, Anal. Chem. 67 (1995) 2883–2891. [5] S.I. Ertel, B.D. Ratner, T.A. Horbett, J. Biomed. Mater. Res. 24 (1990) 1637–1659. [6] D.Y. Tseng, E.R. Edelman, J. Biomed. Mater. Res. 42 (1998) 188–198. [7] H.J. Griesser, R.C. Chatelier, T.R. Gengenbach, G. Johnson, J.G. Steele, J. Biomater. Sci. Polym. Ed. 5 (1994) 531–554. [8] P.A. Ramires, L. Mirenghi, A.R. Romano, F. Palumbo, G. Nicolardi, J. Biomed. Mater. Res. 51 (2000) 535–539. [9] A. Harsch, J. Calderon, R.B. Timmons, G.W. Gross, J. Neurosci. Methods 98 (2000) 135–144. [10] M. Chen, P.O. Zamora, P. Som, L. Pen˜a, S. Osaki, J. Biomater. Sci. Polym. Ed. 14 (2003) 917–935. [11] D.L. Mooradian, P. Trescony, K. Keeney, L.D. Furcht, J. Surg. Res. 53 (1992) 74–81. [12] J.G. Steele, G. Johnson, C. McFarland, B.A. Dalton, T.R. Gengenbach, R.C. Chatelier, P.A. Underwood, H.J. Griesser, J. Biomater. Sci. Polym. Ed. 6 (1994) 511–532. [13] J. Yang, J. Bei, S. Wang, Biomaterials 23 (2002) 2607–2614. [14] T. Pompe, K. Keller, G. Mothes, M. Nitschke, M. Teese, R. Zimmermann, C. Werner, Biomaterials 28 (2007) 28–37. [15] A. Oyane, M. Uchida, Y. Yokoyama, C. Choong, J. Triffitt, A. Ito, J. Biomed. Mater. Res. A 75 (2005) 138–145. [16] D.J. Balazs, K. Triandafillu, P. Wood, Y. Chevolot, C. van Delden, H. Harms, C. Hollenstein, H.J. Mathieu, Biomaterials 25 (2004) 2139–2151. [17] H.U. Lee, Y.S. Jeong, S.Y. Jeong, S.Y. Park, J.S. Bae, H.G. Kim, C.R. Cho, Appl. Surf. Sci. 254 (2008) 5700–5705. [18] I. Gotman, J. Endourol. 11 (1997) 383–389. [19] M. Chen, P.O. Zamora, L. Pen˜a, P. Som, S. Osaki, J. Biomed. Mater. Res. 67A (2003) 994–1000.
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Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Biological response of stainless steel surface modified by N2O/O2 glow discharge plasma Meng Chen *, Shigemasa (Gem) Osaki, Paul O. Zamora BioSurface Engineering Technologies (BioSET), Inc., 9430 Key West Avenue, Rockville, MD 20850, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2008 Received in revised form 18 March 2009 Accepted 24 March 2009 Available online 1 April 2009
Stainless steel wafers were treated with the glow discharge plasma of mixed N2O and O2 at different molar ratios at a certain discharge condition to create desirable biological characteristics to the surfaces. It was found that the molar ratio of N2O to O2 in the mixture at 1:1 used for plasma surface modification caused high apoptotic percentage. Contact angle measurement showed that the surface of stainless steel samples became very hydrophilic after the plasma modification with a value of 158–308. The control stainless steel chips without plasma treatment had a contact angle of 40 28. The data of Electron Spectroscopy for Chemical Analysis (ESCA) indicated that there was a certain amount of oxynitrites formed on the plasma treated surfaces, which was considered to play an important role to cell apoptosis and anti-clot formation in cell culture tests. The ESCA depth profile of up to 250 A˚ from the top surface showed the change of elemental compositions within 40–50 A˚ of the surface by the plasma treatment. The decreased platelet attachment, combined with increased apoptosis in fibroblasts is a distinct combination of biological responses arising from the mixed gas plasma treatment. These initial results suggest it may be of particular use relative to stainless steel stents where decreased platelet attachments are advantageous and induction of apoptosis could limit in-stent restenosis. ß 2009 Elsevier B.V. All rights reserved.
PACS: 52.40.Hf 33.60 Fy 87.14. g Keywords: Surface modification ESCA Nitrous oxide Cell culture Apoptosis Glow discharge plasma
1. Introduction Plasma treatment is a unique technique used to alter biomaterial surface property and its consequent biological response. Glow discharge plasma, known as low temperature plasma, is generated by energizing a gas inside a vacuum chamber. The active components of plasma include ions, electrons, radicals, excited species and photons, among others. The collective properties of these active species can be controlled and harnessed so as to perform a variety of surface treatments, including nanoscale cleaning, activation for surface wettability, chemical grafting, and creating surface bioactivity. Because of the highly reactive environment, plasma can be employed to change the properties of surfaces without affecting the bulk materials. Furthermore, plasma is a dry surface treatment technique, so there are no waste chemicals to dispose of, making this an environmentally friendly process involving very few consumables. Owing to these advantages the plasma surface modification process has been widely used in recent decades for the preparation of biomedical materials
* Corresponding author at: ExcelCoat, LLC, 30670 Ardmore Ct., Novi, MI 48377, United States. E-mail address: [email protected] (M. Chen). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.03.079
with unique surface performances and in the manufacturing of medical devices [1,2]. Oxygen plasmas were used to increase the attachment of cells to polymer surfaces [3–5]. Plasmas were also utilized to introduce amines and amides to polymeric materials for increasing the attachment of cells, and in particular endothelial cells [6–10]. It has also been reported that the plasma treatment resulted in a change in the amount and type of blood proteins that are adsorbed on the treated biomaterial surfaces. For instance, fibronectin and vitronectin are two such blood proteins whose absorption is modified by glow discharge [11,12] thus influencing endothelial cell attachment. Oxygen plasma treatment of polymers was also found conducive to the binding of laminin, another protein that influences cell attachment [11]. Surface properties of poly(D,L-lactide) modified by combining plasma treatment and collagen modification demonstrated considerable improvement in cell affinity [13]. Degradable poly(hydroxybutyrate) thin film treated with low pressure ammonia plasma or low pressure water vapor plasma showed increased hydrophilicity and improved cell adhesion [14]. Another biodegradable polymer, poly(epsiloncaprolactone) (PCL) was modified by an O2 plasma surface treatment to form oxygen-containing functional groups. Consequently, the apatite layer strongly adhered to the PCL surface in the resulting apatite-PCL composite [15]. The surface modification of medical-grade poly(vinyl chloride) (PVC) using a radio-frequency
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oxygen glow discharge pre-functionalization followed by a twostep wet-treatment in sodium hydroxide and silver nitrate solutions was proved to prevent bacterial adhesion to PVC tubes [16]. The surface modification of biocompatible PCL film treated by atmospheric cold plasma with reactive gases was also reported to improve cell distribution and growth [17]. In addition to synthetic polymers, metals like stainless steel and titanium or alloys widely used in the construction of medical devices [18] are also treated with plasmas for a variety of purposes. Plasma treatment of stainless steel and titanium resulted in surfaces with enhanced resistance to platelet and leukocyte attachment [19]. Surface modification of stainless steel by argon plasma pretreatment followed by grafting of poly(ethylene glycol) was found to be very effective in preventing protein adsorption [20]. Surface cleaning and modification of metallic biomaterials using glow discharge plasma treatment provide a clean and reproducible starting condition for further plasma treatments to form well-controlled surface layers [21]. Polished titanium samples were treated using a plasma exposure apparatus in gas atmospheres of air, CO2, and C3F8. It was observed that plasma exposure in a specific gas atmosphere regulated the bonding strength of titanium surface to resin in dental applications [22]. Simvastatin acid (SVA) has been reported to stimulate bone formation. The largest amount of SVA was adsorbed on O2 plasma treated hexamethyldisiloxane (HMDSO) surfaces compared to untreated titanium, HMDSOcoated titanium, and O2 plasma treated titanium [23]. Plasmacoated nitinol, alloy of nickel and titanium, intravascular stents showed increased surface hydrophilicity and enhanced anticoagulation property [24]. The objective of this study is to impart unique biological characteristics to the surface of stainless steel material, which includes decreased platelet attachment and increased apoptosis in fibroblasts. Plasmas of nitrous oxide mixed with oxygen (N2O/O2) at various molar ratios were used to modify the surface of stainless steel chips. The effect of the plasma surface treatment was characterized by means of surface contact angles and Electron Spectroscopy for Chemical Analysis (ESCA). The biological responses of stainless steel before and after the surface modification were evaluated by means of clot formation test and cell apoptosis test of C3H10T1/2 fibroblasts. The correlation between surface functional groups formed from plasma treatment and cell apoptotic event was discussed. 2. Experimental 2.1. Materials
Table 1 Material analysis of 316 stainless steel used in this study. Element
Atomic fraction
Carbon (C) Manganese (Mn) Phosphorus (P) Sulfur (S) Silicon (Si) Chromium (Cr) Nickel (Ni) Nitrogen (N) Copper (Cu) Iron (Fe)
0.08% max. 2.00% max. 0.045% max. 0.030% max. 1.00% max. 16.00–18.00% 10.00–14.00% 0.10% max. 2.00–3.00% Balance
USA), which was 10% of polyethylene glycol based detergent (CAS No.: 9003-11-6) in ethanol, and rinsed exhaustively with water, and air-dried. 2.3. Experimental setup and process parameters The plasma reactor used in this study is shown in Fig. 1. The radio-frequency (13.56 MHz) power supply was applied to plasma chamber through a matching network (not shown in the figure). Between the two high-voltage driven electrodes a discharge zone of 19 cm 16 cm 23 cm (width length height) was established. Prior to feeding of working gases to the plasma chamber, a base vacuum of better than 10 mTorr was achieved in about 5 min using vacuum pumps. The vacuum pumps include one turbomolecular pump (Osaka Vacuum, Model TS433) purchased from Osaka Vacuum, Ltd., San Jose, CA, USA, and one mechanical pump system consisting one rotary vane vacuum pump (Alcatel 2033CP+) and one roots blower (Alcatel RSV 300B) procured from Alcatel Vacuum Products, Fremont, CA, USA. A precision motorized throttle valve was used to regulate the plasma system pressure. The valve was electronically connected to a throttle valve controller. This controller was electronically connected to a main pressure transducer and to a control interface unit which was hooked up with a computer for controlling the operation of the entire plasma system. The computer read the system pressure from the main pressure transducer and compared with the pressure set point sent by the computer. The system feedback loop regulated the opening of throttle valve to maintain the system pressure at the set point pressure constantly. The working gases such as N2O, O2 or NH3 were introduced into the plasma chamber through the inlet located in the front of the chamber. The molar ratios or flow rate ratios of N2O over O2 used in the experiments varied from 0:1, 1:4, 1:2, 2:3, 1:1, 3:2, 4:1 to 1:0. The operational
Square wafers in the dimension of 7.1 mm 7.1 mm 2 mm were used in the studies. They are made of 316 stainless steel (SS), a ‘‘surgical stainless steel’’. This type of stainless steel will not rust or tarnish, is not magnetic, and is generally recognized as nonallergenic. Its elemental composition obtained from the Salem Specialty Ball Company, Canton, CT, is listed in Table 1. The stainless steel sheet, from which the square wafers were cut, was purchased from McMaster-Carr, a supply company at New Brunswick, NJ, USA. Nitrous oxide (N2O) with purity of 99.9995%, VLSI grade, and oxygen (O2) with purity of 99.9%, industry grade, were purchased from Air Products and Chemicals, Inc., Allentown, PA, USA. Ammonia (NH3) with concentration of 99.999%, electronic grade, was also supplied by Air Products and Chemicals, Inc. 2.2. Cleaning process for SS wafers All the SS wafers were cleaned with diluted (2% in distilled water) Acationox detergent (Baxter Healthcare Corp., Deerfield, IL,
Fig. 1. Schematic diagram of plasma reactor used in this study.
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plasma parameters were discharge power of 110 W, working pressure of 50 mTorr, and a total mass flow rate of 50 standard cubic centimeters per minute (sccm) for the gas mixture of N2O/O2 or NH3/O2. In all cases, the plasma treatment time was set at 45 s chosen on the optimal effect of plasma processing on stainless steel surface in regard to N or O elemental composition formed on the surface.
mixing platform in a lateral position (100 cycles/min) for 15 min then transferred to a 37 8C incubator. The mixing and warming were repeated for a total of 1 h. The wafers were rinsed 4 times in buffered saline and fixed in buffered formalin. Images of the wafers were then scanned as digitized images.
2.4. Contact angle measurement
3.1. Contact angle of plasma treated coupons
The contact angle measurement was performed using NRL 10000 Contact Angle Goniometer manufactured by Rame´-hart, Inc., Mountain Lakes, NJ, USA. The HPLC grade distilled water was used for the drops on sample surfaces. All the measurements were carried out at room temperature and the static water contact angle was obtained. Contact angle data were utilized to see how the stainless steel surface was affected by plasma treatment from the perspective of surface hydrophilicity and what correlation it might have to its surface bioactivity.
In Fig. 2, the contact angle data showed that the surface of stainless steel samples became very hydrophilic after plasma modification of N2O/O2 plasma with the average values for n = 5 from 158 to 308. In contrast, the control stainless steel chips without plasma treatment rendered a contact angle of 40 28. The measurement was carried out at 1 week after plasma treatment. Those plasma treated SS wafers were stored in plastic vials until use. The large standard deviations in the contact angle data observed for most conditions could be due to the unstable surface functionality created by NO2/O2 plasma treatment under those conditions. Contact angle analysis is widely used to evaluate the wettability of surface and has been suggested to be a key determinant of cell attachment [25,26]. Wettability is known to affect the adsorption of blood proteins thus regulating a variety of cell behaviors such as cell attachment. The experimental results from Webb et al. [25,26] indicated that contact angles from 20 to 40 rendered the highest anchorage-dependant-cell attachment for the materials they evaluated. In the present study, a highly hydrophilic surface was created on stainless steel by plasma treatment, but a much less blood clot formation, or a reduced level of platelet attachment, was observed on the plasma treated surfaces as compared to untreated controls (see Section 3.4 for blood clot result). This observation implies that the bioactive groups containing oxygen and/or nitrogen formed on the substrate surface other than surface hydrophilicity may play a critical role in platelet attachment and cell apoptosis.
2.5. Surface chemical composition studies ESCA was accessed under contract with the Department of Chemistry, University of Utah, Salt Lake City, UT, USA, in which the ESCA instrumentation was VG Scientific 220i-XL imaging multitechnique surface analysis system, using an Al Ka X-ray source. Material Interface, Inc., Sussex, WI, USA, was another source accessible to this study for performing ESCA measurement. The relevant equipment was Physical Electronics Model 5802 Multitechnique System with a monochromatic aluminum anode and an analysis area of approximately 0.8 mm 2 mm (width length), taking-off angle of 458 with respect to the incident angle of X-ray source. 2.6. Apoptosis test Apoptosis is the carefully regulated process of cell death. The appropriate regulation of apoptosis is important for the prevention of many disease states. In the present study, we use cell apoptotic percent as a criterion to evaluate the efficacy of plasma surface treatment on slowing down the healing process of vessel injuries resulting from implant placement in clinical applications. Plasma treated stainless steel wafers were disinfected, placed in wells of 24-well of low-attachment tissue-culture plates (Corning Inc., Corning, NY, USA), and aliquots of mouse fibroblasts (C3H10T1/2; ATCC, Manassas, VA, USA) added. At selected times the specimens were rinsed, fixed, stained with bis-benzimide (a DNA-binding fluorochrome), and viewed with a fluorescence microscope. Cells were scored as apoptotic if the nuclei were blistered, evidenced condensation of chromatin, and/or evidenced fragmentation of the nucleus.
3. Results and discussion
3.2. ESCA result The survey scan measurement results of ESCA are tabulated in Table 2. The data showed that the elemental composition of both nitrogen (N) and oxygen (O) was increased at the stainless steel surface after N2O/O2 plasma treatment, indicative of oxynitrites functional groups formed on the surface, which was considered to cause the cell apoptosis and decreased platelet adhesion and clot formation. Oxynitrites have been found to promote cellular damage and ultimately cell death by other researchers [27–30]. The elevated O and N component levels on the surface could also be the cause for decreased contact angle. As one derivative of
2.7. Clot formation test In this test, human blood was obtained by venipuncture and collected into specially prepared glass tubes containing a suboptimal amount of heparin (approximately 1/10 normal). This was designed to slow but not to stop clotting. The blood was collected into plastic centrifuge tubes to minimize (but not eliminate) platelet activation. Aliquots of 1 ml of blood were added to 15 ml capacity centrifuge tubes containing stainless steel wafers with or without plasma treatment. The plasma treatment was performed using gas mixture of N2O/O2 (12.5:12.5) for 45 s. To each tube had been added 4 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA, USA). The capped tubes were mixed on a
Fig. 2. Contact angle of control and plasma treated SS chips versus different ratios of N2O to O2. The data are presented as means standard deviations for n = 5.
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Table 2 Surface composition as determined by Electron Spectroscopy for Chemical Analysis (ESCA) of stainless steel wafers: control and N2O/O2 plasma treated (at molar ratio of 1:1). Sample identification
None N2O/O2
Atomic fraction for elements with 50 A˚ of the SS surface C
N
O
F
Si
S
Cl
Cr
Fe
Ni
Mo
21.9 17.0
0.5 1.3
52.5 57.9
0.6 0.4
1.9 3.5
1.0 0.1
0.2 0.0
10.7 7.2
9.5 10.4
1.1 1.9
0.2 0.3
oxynitrites, nitric oxide (NO) on the surface is known to have potent anti-platelet aggregating activity [31–37]. Nitric oxide (NO) release polymers to prevent thrombus formation have been found to cause a dramatic decrease in platelet adhesion and surface clot formation (compared to blank films) during both in vitro and in vivo experiments [37]. It is not clear how the carbon content at the substrate surface affects its bioactivity. To further study the potential influence of carbon at surface on cell apoptosis or other biological responses, a hydrogen plasma treatment will be applied to stainless steel substrates in follow-on studies since it could provide a more pristine surface with reduced carbon content. The trace amount of fluorine detected at the surface of both untreated and N2O/O2 plasma treated specimens could be attributed to the background noise from ESCA measurement. Depth profiling of the main elements including C, N, O, Si, Cr, Fe, and Ni for both untreated and treated SS wafers was plotted in Fig. 3. The SS samples were depth-profiled using ESCA with an argon ion beam at 20 A˚/min versus SiO2. Notably, the atomic fractions of C, N and Si for both plasma treated and untreated SS were around 1–2% in the near surface layer and did not exhibit difference between treated and untreated ones due to their low values. A deeper iron depleted surface layer (100 A˚) was observed for the N2O/O2 plasma treated SS sample than that (60 A˚) of the untreated control. In the case of plasma treated sample, the oxygen depth profile was broader than that for untreated control. The depth profile of the Cr and Ni of
plasma treated sample was shifted toward the inside too, which could be a result of oxygen incorporation in the near surface layer during the surface modification process using N2O/O2 plasma. These changes observed in the elemental components revealed that the N2O/O2 plasma surface treatment could modify the stainless steel surface to a depth of about 40–50 A˚ from the top surface generating a thin layer of oxygen-containing functional groups, such as oxynitrites, other than metal oxides. 3.3. Apoptosis The apoptotic percent of cells is plotted in Fig. 4 versus the molar ratio of N2O/O2. The untreated wafer is included as control, and the result of doxorubicin used as a positive control is also presented. It is found that the ratio of 1:1 (actual mass flow rate of N2O to O2 is 12.5 to 12.5 sccm) of N2O to O2 in the mixture resulted in the highest apoptotic percent in the cell culture experiments. Please note, the original data of the apoptotic percentage indicated that for each condition with two determinations the variation was less than or equal to 1%. Therefore, it is reasonable to believe that more than 1% apoptotic percentage difference from sample to sample would be significant. Fig. 4 also indicated that ammonia/ oxygen (NH3/O2) plasma treated stainless steel surface exhibited a similar increase in apoptosis to N2O/O2 (at the ratio of 1:1) plasma modified surface suggesting that this property may be common to
Fig. 3. Comparison of depth profiles for stainless steel chips with or without N2O/O2 Plasma treatment. Samples were depth-profiled using ESCA with an argon ion beam at 20 A˚/min versus SiO2.
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Fig. 4. Apoptosis in C3H10T1/2 fibroblasts at 1 day after seeding on stainless steel wafers with and without plasma treatment. Notes: control—no treatment, am-ox— ammonia/oxygen plasma, and doxorbn stands for doxorubicin which is used as a positive control, (1) N2O (0)/O2 (25), (2) N2O (5)/O2 (20), (3) N2O (8)/O2 (16), (4) N2O (10)/O2 (15), (5) N2O (12.5)/O2 (12.5), (6) N2O (15)/O2 (10), (7) N2O (20)/O2 (5), and (8) N2O (25)/O2 (0). The data are the average of two determinations with both of the values closely agreeing.
surfaces coated with oxynitrites. Actually it was found that oxynitrites were formed on NH3/O2 plasma treated stainless steel surfaces based on the ESCA data that had been reported elsewhere [19]. We also performed ESCA analysis on all the stainless steel samples treated with plasmas at different molar ratios of N2O to O2 as presented in Fig. 4. However, no clear correlation could be established between the elemental compositions of N and O and the corresponding apoptosis data for all the molar ratios used. As a result, we focused on the unique condition of N2O to O2 at the ratio of 1:1 in an effort to better study and understand the surface chemistry created by this plasma treatment and the plausible mechanism of its generating high apoptosis on the treated surface. To treat the major health problem in the United States, that is coronary heart disease, stenting has been widely accepted to reduce restenosis rate after percutaneous balloon angioplasty. The coronary artery stent, a small mesh tube made of stainless steel or other metal or alloys, functions as a scaffold to prop open blocked heart arteries to keep the passages from reclosing. However, the restenosis remains as an issue with stenting to some patients since the biological response to the vessel damage caused by stent implantation is stimulation of accelerated growth of arterial smooth muscle cells. Creating smooth muscle cell apoptosis to an adequate level on the surface of stainless steel is considered as an effective approach to inhibit cell proliferation and prevent rapid healing of tissue around the stents made of stainless steel. 3.4. Clot formation Fig. 5 is the image of stainless steel wafers after blood clot test. Apparently, the wafers without plasma treatment exhibited blood clot, whereas the treatment of stainless steel with nitrous oxide/ oxygen (N2O/O2) plasma resulted in resistance to clot formation in vitro. This result indicated that the level of platelet attachment to stainless steel following treatment with the N2O/O2 plasma was reduced considerably as compared to untreated controls. Also shown in Fig. 5 is a reduced clot formation on the SS wafers treated with plasma of NH3 and O2, suggesting that other combinations of nitrogen-containing gases plus oxygen or oxygen-containing gases could mimic the reduced clot formation due to the formation of similar functional groups such as bioactive oxynitrites on the substrate surface. The metal atoms on the substrate were found not critical in decreasing platelet attachment, since stainless steel
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Fig. 5. Treatment of stainless steel with either ammonia/oxygen plasma or nitrous oxide/oxygen plasma resulted in resistance to clot formation in vitro.
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