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Surface modification of polyurethane and silicone for therapeutic medical technics by means of electron beam
Surface & Coatings Technology 205 (2010) 1618–1623
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Surface & Coatings Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t
Surface modification of polyurethane and silicone for therapeutic medical technics by means of electron beam C. Wetzel a,⁎, J. Schönfelder a, W. Schwarz a, R.H.W. Funk b a b
Fraunhofer Institute for Electron Beam and Plasma Technology (FEP), Dresden, Germany University of Technology Dresden, Institute of Anatomy, Dresden, Germany
a r t i c l e
i n f o
Available online 12 August 2010 Keywords: Electron beam Biofunctionalization Surface modification Silicone Polyurethane Medical technics
a b s t r a c t Surface modification technologies are gaining growing acceptance for treatment of implant materials to enhance biocompatibility. Our examinations focus on polyetherurethane and silicones, two typical flexible implant materials, which we have modified by non-thermal electron beam processing. Advantages of this method are the adjustable degree of modification as well as the simultaneous sterilizing effects. The polymer surfaces were characterized with regard to wetting behavior, surface energy, chemistry and morphology. The cell adhesion was examined too. The results reveal that the electron beam is a useful tool for surface modification of polymers. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Medical products that are used for applications on or in the human body must be made of materials which are fully compatible with the surroundings at the point of application. This puts high requirements on materials, and on polymers in particular, many of which are not sufficiently biocompatible in their as-produced state [1]. In order to enhance the biocompatibility, and in particular the biofunctionality of materials used for implants, increasing use is being made of surface modification techniques [2,3]. The use of electron beam technology for surface modification has, however, been little reported, even though this approach has many advantages. Particular benefits worthy of mention here are the ability to adjust the degree of modification like cleavage of bonds, reticulation and insertion of new chemical groups as well as a simultaneous sterilization of the surface [4]. With careful choice of specific parameters, it is possible to treat sensitive substrates such as biomaterials under gentle conditions [5]. Treatment by low-energy electrons is able to modify the surface of a material without adversely affecting the properties of the base material. The electron beam, mostly secondary electrons, excites and ionizes atoms and polymer molecules on the targeted substrate surface and the penetrated random layer. Atoms and molecules of surrounding media (atmosphere and water) are included in these reactions. Radicals, produced on this way, promote the decomposition and/or crosslinking of the polymer surface as well as implantation of new functional groups, which depends on present media during the
⁎ Corresponding author. Tel.: + 49 351 2586 165; fax: + 49 351 2586 55 165. E-mail address: [email protected] (C. Wetzel). 0257-8972/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2010.07.103
treatment. The resulting network density is affected by availability of unconsumed radicals from the atmosphere which participate in crosslinking reactions and by the quantity of chain scission reactions which are in time-dependence on exposure. Related to experimental conditions all reactions which are initiated and take part in the treatment process are in concurrence to each other. The summary of them delivers the final product modification. The modification of polyurethane and silicone polymers by electron beam treatment is presented in this paper. The treated surfaces were subsequently characterized for their hydrophilicity, surface energy, chemical composition, and morphology. In vitro tests using mouse fibroblasts (L929) demonstrated the improved biofunctionality of polyurethane after electron beam treatment. No fibroblasts adhered to either untreated silicone or to silicone treated with an electron beam. However, the material appeared to be protected against damage caused by cells and their enzymes as a result of the modification of the surface molecules. The results show that electron beam technology is a suitable method for functionalizing polymer surfaces for use in therapeutic medical products [6].
2. Experiments 2.1. Electron beam treatment In general, electrons accelerated with low energy between 100 and 150 kV, are used for the treatment. No radioactivity is present and X-ray shielding is not too expensive. Modern units are available by low cost. One advantage is the exact guidance of applied energy doses in (mostly short) time, amount and penetration depth. The
C. Wetzel et al. / Surface & Coatings Technology 205 (2010) 1618–1623
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For the biofunctionalization, the electron beam treatment is applied at atmospheric pressure. The accelerated electron beam passes from the high vacuum generation chamber through a beam exit window, a thin metal foil, to atmosphere in an X-ray shielded reaction chamber. For the treatment of the polyurethane and silicone samples, the electron emitter was set in our experiments to an acceleration voltage of 150 kV and an electron current outlet of 10 mA. Hereby the effective penetration depth into common polymer surfaces and boundary layers is about 100 μm [8,9]. The treatment was carried out in an air atmosphere at normal pressure. Samples were linear carried below the electron beamer and exposed to irradiation. The total dose applied to the samples amounted to up to 6000 kGy. High doses were realized by subsequent irradiation steps in single doses of 100 kGy with pauses of 1 min to prevent overheating of the polymer material [10].
2.2. Polymer characterization The contact angle measurements were also carried out using the contact angle test unit (OCA20) and SCA22 software (version 3.12.11) from Dataphysics Instruments GmbH (Filderstadt). The “sessile drop” method was used. Via an electronically controlled injection module, a 1 μl droplet of the test liquid (deionized water or ethylene glycol) was applied to the test surface at an ambient temperature of ca. 21 °C in air atmosphere. After manual determination of the baseline of the photographed droplet, the contact angle was measured automatically by the image recognition software in the unit. Usually the method according to Owens–Wendt–Rabel–Kaelble (OWRK) is applied for calculation and was used here [11,12]. The ATR-FTIR spectra were taken on a Spectrum 2000 instrument (Perkin Elmer GmbH) using a compatible ATR module with germanium crystal (Specac Inc.). The spectra were recorded in the region from 700 cm− 1 to 4000 cm− 1 at a resolution of 4 cm− 1. Examinations by X-ray photoelectron spectroscopy (XPS) were made at UHV XPS/SPM System of Omicron Nanotechnology GmbH. This established method uses an X-ray beam, directed to the sample surface. The impact energy of photoelectrons is absorbed by core electrons of the elements. If the energy high enough resp. is equal to binding energy (unique for every element), the electrons emit out of surface. By analysis of their energy and intensity the presence and composition of chemical elements on the surface can be detected: especially here the C 1 s, Si 2p or O 1 s lines [13,14].
Fig. 1. ATR-FTIR spectra of PUR in the wavelength range 3600–1000 cm− 1 (both above) and of silicone in the wavelength range 1300–900 cm− 1 (below).
quantity of absorbed energy per unit of mass of treated material is expressed in Gray (Gy): 1 Gy = 1 Joule/kg [7]. Compared to most other modification methods, the electron beam not only functionalizes the surface but also simultaneously sterilizes it [4].
Fig. 2. Contact angle with water of polyurethane and silicone as a function of the electron beam dose.
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Fig. 3. Surface energy and polar contribution of surface energy on polyurethane and silicone as a function of the electron beam dose.
The polymer surface hardness was measured using a Nano Indenter XP (MTS). Tensile tests were carried out on an UPM 1456 unit manufactured by Zwick, Ulm, with rectangle samples (width 20 mm, length of restraint 100 mm). The elongation of test strips at fracture was measured and the average of five specimens has been taken. 2.3. In vitro tests The biofunctionality of the materials was evaluated with the help of the cell proliferation test WST-1. Using this method, quantitative statements can be made about adherent active cells on the material surface. The tests involved incubating fibroblasts from connective tissue from mice (L929) for four days on the samples. Using the untreated and electron beam treated (1000 kGy) samples, the staining of the cell nuclei (DAPI staining) allowed the cells to be counted under the fluorescence microscope (BX60F5, Olympus) [16]. 3. Results 3.1. Characterization of the polymers Fig. 1 demonstrates the changes that were observed in the ATRFTIR spectra of polyurethane and silicone. In polyurethane (PUR), the
Fig. 4. XPS results on untreated polyurethane and on two modified polyurethane surfaces.
Fig. 5. Elongation at fracture of polyurethane as a function of the electron beam dose (kGy).
height of the H-bonded NH absorption band (3320 cm− 1) increased with increasing dose. In contrast, the height of the CH2 bands (3000– 2800 cm− 1) decreased. The increase in the absorption of C = O at 1728 cm− 1 and the formation of new bands at 1780 cm− 1 and 1176 cm− 1 are due to ester formation and to formiates, whereby the C–O–C bond is cleaved (peak at 1110 cm− 1) [17,18]. At 1630 cm− 1 there is a new band, assigned to the formation of primary amines [17]. These changes are due to the decomposition of the main chain of the polyurethane. In addition to the aforementioned effects, the N–C bond is cleaved and this leads to the formation of primary amines, carboxylic acids, and also esters [17,18]. The ATR-FTIR spectrum of silicone also shows evidence of cleavage of the main chain. The Si–O band at 1080 cm− 1 decreases with increasing dose. Fig. 2 shows the contact angle of polyurethane and silicone as a function of the electron beam dose. For both materials, the contact angle decreases systematically with increasing dose. For values up to 6000 kGy, the decrease is ca. 30%. There is a correlation for polyurethane between the measured surface energy and the polar contributions on the surface (Fig. 3). The results for silicone are not clear, due to concurring decomposition and oxidation effects. In principle these effects lead to lower molecular weight surface-active
Fig. 6. Hardness of the silicone surface as a function of the electron beam dose (kGy).
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thane as a function of the electron beam dose. The same findings are known for silicone rubbers according to the duration of plasma treatments [13–15]. The mechanical properties of the polymers can be significantly affected by the electron beam treatment, as can be clearly seen in Fig. 5 for the elongation at fracture of polyurethane. The hardness of silicone increased at high electron beam doses, as demonstrated by hardness measurements (Fig. 6). This result and the causes thereof are comparable to the results of other plasma treatment methods at air. Electron beam treatment (in air) of silicone elastomer surfaces causes in initial jump the replacement of methyl groups by oxygen, decreased total carbon percentage and a silica-like surface layer after long exposure. During radiolysis some evolution of gas, especially methane, is known [1,7,13,14]. 3.2. In vitro tests The biofunctionality of the surfaces was evaluated by fluorescence microscopy. The untreated polyurethane surface had a count of 2380 cells/mm2, whilst there were only 560 cells/mm2 on the treated sample (1000 kGy). Fig. 7a and b show micrographs for this. The results were quantitatively confirmed using the cell proliferation test WST-1 (Fig. 8). Adhesion of active fibroblasts on polyurethane decreased considerably with increasing electron beam dose. At a dose of 3000 kGy, only 5% of the originally adhering cells were present. No cells adhere to the silicone surfaces — either untreated or treated. Despite this, electron beam treatment still
Fig. 7. a. DAPI stained cell nuclei on an untreated polyurethane surface (average value from in each case 5 measurements. Magnification: 400-fold). b. DAPI stained cell nuclei on a treated polyurethane surface (1000 kGy) (average value from in each case 5 measurements. Magnification: 400-fold).
species on polymers, yet these cannot be specifically identified on silicone [19]. XPS measurements complement these findings. Fig. 4 shows lowered carbon, but marked increased oxygen content in polyure-
Fig. 8. Adhesion of fibroblasts (L929) on PUR, studied using WST-1 assay (average value from 5 measurements).
Fig. 9. a. Morphology of untreated silicone after 6 week exposure to cells and cell culture medium. b. Morphology of treated silicone (1000 kGy) after 6 week exposure to cells and cell culture medium (C: untreated, EB: treated with electron beam, P: treated with plasma) [7].
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Fig. 10. Surface energy (mN/m) of polyurethane.
brought a benefit. The material is not attacked by the cells and their enzymes. In each case 5 test series were measured over a 6 week time cycle. Damage was clearly apparent on the untreated samples (Fig. 9a) but there were no changes, however, on the samples treated with electron beam (Fig. 9b).
4. Discussion A possible cause of the reduced attack on silicone surfaces could be the reticulation reactions which take place during enhanced treatment conditions. The substitution of methyl groups by oxygen leads to more Si–O–Si bonds which are known to be resistant to breakage in biochemical environment (biohydrolysis). This reticulation, finally to a three dimensional network, effects a greater resistance to degradation resp. decomposition induced by cells and the cell culture medium. Most degradation, so is reported, causes lipid absorption and altering the nature of filler–binder interactions play a major role too [20,21]. In addition, the initially very weak material gains strength due to the electron beam treatment. As in original silicones (e.g. tubes) are particularly soft materials, their mechanical properties can be significantly improved by customized electron beam treatment. For
each area of application of silicone materials, optimum treatment parameters for the material properties must be determined. The contact angle, surface energy, and number of polar surface groups are key parameters when discussing wetting phenomena, because when two phases come into contact with each other only similar groups interact. The increase in the polar groups is caused by reactions of oxygen with polymer molecule radicals which simultaneously result in the lowering of the contact angle [22]. The oxygen content on the surface arises very fast just at the beginning of the treatment [14]. Comparative studies by the authors using plasma treatment and electron beam treatment resulted in a much greater increase in the surface energy for plasma treatment, and in particular of the polar contributions (Fig. 10), [16,23,24]. Analogously, the cell adhesion tests showed a factor of 4 higher number of fibroblasts. In general it was established that the fibroblasts proliferate to a greater extent on surfaces having a low contact angle (Fig. 11). Other literature also reports that cell adhesion is higher on hydrophilic, energy-rich surfaces [22,24,25]. This trend could only be confirmed to a limited extent via the electron beam treatment. Basically, it was found that the adhesion of the fibroblasts drastically decreased even though the hydrophilicity and the surface energy slightly increased. This phenomenon has also been observed by other authors [26,27] and not only indicates the complexity of the treatment, but also opens the door for many potential applications. There is still no absolute clarity about the exact mechanism of cell adhesion. There is agreement, however, that not only the surface energy, roughness, texture and charge affect cell adhesion, but also chemical reactions of the surface groups, crystallinity and mechanical properties which can promote or hinder the adhesion [28–31]. The magnitude of such effects of individual parameters on the adhesion and which role the combination of specific parameters does play still must be determined. There are applications of the experimental results the authors see in the therapeutically medical technics with polymer products. The functionality can be perfectly adapted for cell adherents and also for chemical and physical properties with “surface-gradient-layers”. 5. Conclusions These studies on polyurethane and silicone have shown that electron beam treatment is a suitable tool for modifying polymers. The electron beam treatment of polyurethane resulted in lower adhesion of fibroblasts (L929). No fibroblasts adhered to silicone — either untreated or electron beam treated. However, the resulted modification of the surface molecules appears to protect the material against damage by the cells and their enzymes. The higher stiffness caused by the electron beam treatment – for equivalent implant geometry – is however very important for adapting medical products and could provide an improved “stress shielding” effect. The application-specific optimization of the biofunctionality and the simultaneous sterilizing effect make the electron beam treatment a very promising method for a wide range of uses. The aim of ongoing further work is the identification of parameters for specific applications, so opening up a number of applications in therapeutic medical technics. The authors thank Steffen Strehle (University of Technology, Dresden) and Konrad Schneider (Leibniz Institute of Polymer Research, Dresden) for their assistance in XPS and mechanical tests. References
Fig. 11. Proliferation rate as a function of the contact angle of polyurethane.
[1] E. Wintermantel, S.W. Ha, Medizintechnik — Life Science Engineering, 4th edn, Springer, Berlin-Heidelberg, ISBN: 978-3-540-74924-0, 2008. [2] M. Epple, Teubner, Wiesbaden (2003) 49. [3] L.L. Hench, E.C. Ethridge, Biomaterials — An Interfacial Approach, Academic Press, New. York, 2004. [4] E. Gautriaud, K.T. Stafford, J. Adamchuk et al., BioProcess International, Suppl., Chapt.
C. Wetzel et al. / Surface & Coatings Technology 205 (2010) 1618–1623 [5] H. Planck, Kunststoffe und Elastomere in der Medizin, W. Kohlhammer, Stuttgart, , 1993. [6] C. Wetzel, N. Ozkucur, J. Schoenfelder, T.K. Monsees, R.H.W. Funk, 1st Biomat.Africa, Pretoria, 2009. [7] A. Chapiro, Radiation Chemistry of Polymeric Systems, High Polymers, Vol XV, Wiley&Sons, New York-London, 1962, p. 473. [8] K. Mehta, I. Janovsky, Radiat. Phys. Chem. 47 (3) (1996). [9] V. Lazurik, V. Moskvin, T. Tabata, IEE Trans. Nucl. Sci. 45 (3) (1998) 626. [10] F-H. Roegner, C. Wetzel, O. Roeder, G. Gotzmann, SVC 52nd Ann. Tech. Conf., Santa. [11] Group of authors, Guide for Contact Angle Measurement, DataPhysics Instruments. GmbH, Filderstadt, 2006. [12] H.-D. Dörfler, Grenzflächen und kolloid-disperse Systeme, Springer, Heidelberg (2002) ISBN 3-540-42547-0. [13] D.J. O'Connor, B.A. Sexton, R.St.C. Smart,, Surface Analysis Method in Materials Science, Springer, Heidelberg, 1992; B. Olander, A. Wirsen, J. Appl. Polym. Sci. 91 (2004) 4098. [14] I.F. Husein, Ch. Can, P.K. Chu, J. Mater. Sci. Lett. 19 (2000) 1883. [15] T. Kobayashi, R. Katou, T. Yokota, et al., Surf. Coat. Techn. 201 (2007) 8039.
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[16] J. Schoenfelder, N. Ozkucur, R. Funk, C. Wetzel, Neues Dresdn. Vakuumtechn. Kolloq., 16, 2008. [17] A. Dannoux, et al., Nucl. Instr. Meth. Phys., 236, 2005, p. 488. [18] C. Guignot, et al., Nucl. Instr. Meth. Phys. 185 (2001) 178. [19] H.J. Griesser HJ, et al., J. Biomater. Sci. Polym. Edn. 5 (1994) 531. [20] M.J. Whitford, Biomat. 5 (1984) 298. [21] T.C. Ward, J.T. Perry, J. Biomed. Mat. Res., 15, 1981. [22] E.M. Liston, L. Martinu, W.R. Wertheimer, J. Adh. Sci. Technol. 7 (10) (1993) 1091. [23] C. Wetzel, N. Ozkucur, J. Schönfelder, T.K. Monsees, R.H.W. Funk, Europ. Cells Mat. (ISSN: 1473-2262) 19 (2010) 15 Suppl. 1,. [24] C. Wetzel, O. Roeder, Galvanotechnik 10 (2009) 2244. [25] S. Kothari, et al., J. Mater. Sci. 6 (1995) 695. [26] J.M. Schakenraad, et al., J. Biomed. Mater. Res. 20 (1986) 773. [27] S.B. Kennedy, et al., Biomater. 27 (2006) 3817. [28] L. Marinucci, et al., Int. J. Oral & Maxillofacial Implants 21 (5) (2006) 719. [29] S. Britland, et al., Exp. Cell Res. 198 (1) (1992) 124. [30] M. Lupu, et al., Polym. Int. 56 (2007) 389. [31] E.T. den Braber, J.E. de Ruijter, H.T.J. Smits, et al., J. Biomed. Mater. Res. 29 (1995) 511.
Contents lists available at ScienceDirect
Surface & Coatings Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t
Surface modification of polyurethane and silicone for therapeutic medical technics by means of electron beam C. Wetzel a,⁎, J. Schönfelder a, W. Schwarz a, R.H.W. Funk b a b
Fraunhofer Institute for Electron Beam and Plasma Technology (FEP), Dresden, Germany University of Technology Dresden, Institute of Anatomy, Dresden, Germany
a r t i c l e
i n f o
Available online 12 August 2010 Keywords: Electron beam Biofunctionalization Surface modification Silicone Polyurethane Medical technics
a b s t r a c t Surface modification technologies are gaining growing acceptance for treatment of implant materials to enhance biocompatibility. Our examinations focus on polyetherurethane and silicones, two typical flexible implant materials, which we have modified by non-thermal electron beam processing. Advantages of this method are the adjustable degree of modification as well as the simultaneous sterilizing effects. The polymer surfaces were characterized with regard to wetting behavior, surface energy, chemistry and morphology. The cell adhesion was examined too. The results reveal that the electron beam is a useful tool for surface modification of polymers. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Medical products that are used for applications on or in the human body must be made of materials which are fully compatible with the surroundings at the point of application. This puts high requirements on materials, and on polymers in particular, many of which are not sufficiently biocompatible in their as-produced state [1]. In order to enhance the biocompatibility, and in particular the biofunctionality of materials used for implants, increasing use is being made of surface modification techniques [2,3]. The use of electron beam technology for surface modification has, however, been little reported, even though this approach has many advantages. Particular benefits worthy of mention here are the ability to adjust the degree of modification like cleavage of bonds, reticulation and insertion of new chemical groups as well as a simultaneous sterilization of the surface [4]. With careful choice of specific parameters, it is possible to treat sensitive substrates such as biomaterials under gentle conditions [5]. Treatment by low-energy electrons is able to modify the surface of a material without adversely affecting the properties of the base material. The electron beam, mostly secondary electrons, excites and ionizes atoms and polymer molecules on the targeted substrate surface and the penetrated random layer. Atoms and molecules of surrounding media (atmosphere and water) are included in these reactions. Radicals, produced on this way, promote the decomposition and/or crosslinking of the polymer surface as well as implantation of new functional groups, which depends on present media during the
⁎ Corresponding author. Tel.: + 49 351 2586 165; fax: + 49 351 2586 55 165. E-mail address: [email protected] (C. Wetzel). 0257-8972/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2010.07.103
treatment. The resulting network density is affected by availability of unconsumed radicals from the atmosphere which participate in crosslinking reactions and by the quantity of chain scission reactions which are in time-dependence on exposure. Related to experimental conditions all reactions which are initiated and take part in the treatment process are in concurrence to each other. The summary of them delivers the final product modification. The modification of polyurethane and silicone polymers by electron beam treatment is presented in this paper. The treated surfaces were subsequently characterized for their hydrophilicity, surface energy, chemical composition, and morphology. In vitro tests using mouse fibroblasts (L929) demonstrated the improved biofunctionality of polyurethane after electron beam treatment. No fibroblasts adhered to either untreated silicone or to silicone treated with an electron beam. However, the material appeared to be protected against damage caused by cells and their enzymes as a result of the modification of the surface molecules. The results show that electron beam technology is a suitable method for functionalizing polymer surfaces for use in therapeutic medical products [6].
2. Experiments 2.1. Electron beam treatment In general, electrons accelerated with low energy between 100 and 150 kV, are used for the treatment. No radioactivity is present and X-ray shielding is not too expensive. Modern units are available by low cost. One advantage is the exact guidance of applied energy doses in (mostly short) time, amount and penetration depth. The
C. Wetzel et al. / Surface & Coatings Technology 205 (2010) 1618–1623
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For the biofunctionalization, the electron beam treatment is applied at atmospheric pressure. The accelerated electron beam passes from the high vacuum generation chamber through a beam exit window, a thin metal foil, to atmosphere in an X-ray shielded reaction chamber. For the treatment of the polyurethane and silicone samples, the electron emitter was set in our experiments to an acceleration voltage of 150 kV and an electron current outlet of 10 mA. Hereby the effective penetration depth into common polymer surfaces and boundary layers is about 100 μm [8,9]. The treatment was carried out in an air atmosphere at normal pressure. Samples were linear carried below the electron beamer and exposed to irradiation. The total dose applied to the samples amounted to up to 6000 kGy. High doses were realized by subsequent irradiation steps in single doses of 100 kGy with pauses of 1 min to prevent overheating of the polymer material [10].
2.2. Polymer characterization The contact angle measurements were also carried out using the contact angle test unit (OCA20) and SCA22 software (version 3.12.11) from Dataphysics Instruments GmbH (Filderstadt). The “sessile drop” method was used. Via an electronically controlled injection module, a 1 μl droplet of the test liquid (deionized water or ethylene glycol) was applied to the test surface at an ambient temperature of ca. 21 °C in air atmosphere. After manual determination of the baseline of the photographed droplet, the contact angle was measured automatically by the image recognition software in the unit. Usually the method according to Owens–Wendt–Rabel–Kaelble (OWRK) is applied for calculation and was used here [11,12]. The ATR-FTIR spectra were taken on a Spectrum 2000 instrument (Perkin Elmer GmbH) using a compatible ATR module with germanium crystal (Specac Inc.). The spectra were recorded in the region from 700 cm− 1 to 4000 cm− 1 at a resolution of 4 cm− 1. Examinations by X-ray photoelectron spectroscopy (XPS) were made at UHV XPS/SPM System of Omicron Nanotechnology GmbH. This established method uses an X-ray beam, directed to the sample surface. The impact energy of photoelectrons is absorbed by core electrons of the elements. If the energy high enough resp. is equal to binding energy (unique for every element), the electrons emit out of surface. By analysis of their energy and intensity the presence and composition of chemical elements on the surface can be detected: especially here the C 1 s, Si 2p or O 1 s lines [13,14].
Fig. 1. ATR-FTIR spectra of PUR in the wavelength range 3600–1000 cm− 1 (both above) and of silicone in the wavelength range 1300–900 cm− 1 (below).
quantity of absorbed energy per unit of mass of treated material is expressed in Gray (Gy): 1 Gy = 1 Joule/kg [7]. Compared to most other modification methods, the electron beam not only functionalizes the surface but also simultaneously sterilizes it [4].
Fig. 2. Contact angle with water of polyurethane and silicone as a function of the electron beam dose.
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Fig. 3. Surface energy and polar contribution of surface energy on polyurethane and silicone as a function of the electron beam dose.
The polymer surface hardness was measured using a Nano Indenter XP (MTS). Tensile tests were carried out on an UPM 1456 unit manufactured by Zwick, Ulm, with rectangle samples (width 20 mm, length of restraint 100 mm). The elongation of test strips at fracture was measured and the average of five specimens has been taken. 2.3. In vitro tests The biofunctionality of the materials was evaluated with the help of the cell proliferation test WST-1. Using this method, quantitative statements can be made about adherent active cells on the material surface. The tests involved incubating fibroblasts from connective tissue from mice (L929) for four days on the samples. Using the untreated and electron beam treated (1000 kGy) samples, the staining of the cell nuclei (DAPI staining) allowed the cells to be counted under the fluorescence microscope (BX60F5, Olympus) [16]. 3. Results 3.1. Characterization of the polymers Fig. 1 demonstrates the changes that were observed in the ATRFTIR spectra of polyurethane and silicone. In polyurethane (PUR), the
Fig. 4. XPS results on untreated polyurethane and on two modified polyurethane surfaces.
Fig. 5. Elongation at fracture of polyurethane as a function of the electron beam dose (kGy).
height of the H-bonded NH absorption band (3320 cm− 1) increased with increasing dose. In contrast, the height of the CH2 bands (3000– 2800 cm− 1) decreased. The increase in the absorption of C = O at 1728 cm− 1 and the formation of new bands at 1780 cm− 1 and 1176 cm− 1 are due to ester formation and to formiates, whereby the C–O–C bond is cleaved (peak at 1110 cm− 1) [17,18]. At 1630 cm− 1 there is a new band, assigned to the formation of primary amines [17]. These changes are due to the decomposition of the main chain of the polyurethane. In addition to the aforementioned effects, the N–C bond is cleaved and this leads to the formation of primary amines, carboxylic acids, and also esters [17,18]. The ATR-FTIR spectrum of silicone also shows evidence of cleavage of the main chain. The Si–O band at 1080 cm− 1 decreases with increasing dose. Fig. 2 shows the contact angle of polyurethane and silicone as a function of the electron beam dose. For both materials, the contact angle decreases systematically with increasing dose. For values up to 6000 kGy, the decrease is ca. 30%. There is a correlation for polyurethane between the measured surface energy and the polar contributions on the surface (Fig. 3). The results for silicone are not clear, due to concurring decomposition and oxidation effects. In principle these effects lead to lower molecular weight surface-active
Fig. 6. Hardness of the silicone surface as a function of the electron beam dose (kGy).
C. Wetzel et al. / Surface & Coatings Technology 205 (2010) 1618–1623
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thane as a function of the electron beam dose. The same findings are known for silicone rubbers according to the duration of plasma treatments [13–15]. The mechanical properties of the polymers can be significantly affected by the electron beam treatment, as can be clearly seen in Fig. 5 for the elongation at fracture of polyurethane. The hardness of silicone increased at high electron beam doses, as demonstrated by hardness measurements (Fig. 6). This result and the causes thereof are comparable to the results of other plasma treatment methods at air. Electron beam treatment (in air) of silicone elastomer surfaces causes in initial jump the replacement of methyl groups by oxygen, decreased total carbon percentage and a silica-like surface layer after long exposure. During radiolysis some evolution of gas, especially methane, is known [1,7,13,14]. 3.2. In vitro tests The biofunctionality of the surfaces was evaluated by fluorescence microscopy. The untreated polyurethane surface had a count of 2380 cells/mm2, whilst there were only 560 cells/mm2 on the treated sample (1000 kGy). Fig. 7a and b show micrographs for this. The results were quantitatively confirmed using the cell proliferation test WST-1 (Fig. 8). Adhesion of active fibroblasts on polyurethane decreased considerably with increasing electron beam dose. At a dose of 3000 kGy, only 5% of the originally adhering cells were present. No cells adhere to the silicone surfaces — either untreated or treated. Despite this, electron beam treatment still
Fig. 7. a. DAPI stained cell nuclei on an untreated polyurethane surface (average value from in each case 5 measurements. Magnification: 400-fold). b. DAPI stained cell nuclei on a treated polyurethane surface (1000 kGy) (average value from in each case 5 measurements. Magnification: 400-fold).
species on polymers, yet these cannot be specifically identified on silicone [19]. XPS measurements complement these findings. Fig. 4 shows lowered carbon, but marked increased oxygen content in polyure-
Fig. 8. Adhesion of fibroblasts (L929) on PUR, studied using WST-1 assay (average value from 5 measurements).
Fig. 9. a. Morphology of untreated silicone after 6 week exposure to cells and cell culture medium. b. Morphology of treated silicone (1000 kGy) after 6 week exposure to cells and cell culture medium (C: untreated, EB: treated with electron beam, P: treated with plasma) [7].
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Fig. 10. Surface energy (mN/m) of polyurethane.
brought a benefit. The material is not attacked by the cells and their enzymes. In each case 5 test series were measured over a 6 week time cycle. Damage was clearly apparent on the untreated samples (Fig. 9a) but there were no changes, however, on the samples treated with electron beam (Fig. 9b).
4. Discussion A possible cause of the reduced attack on silicone surfaces could be the reticulation reactions which take place during enhanced treatment conditions. The substitution of methyl groups by oxygen leads to more Si–O–Si bonds which are known to be resistant to breakage in biochemical environment (biohydrolysis). This reticulation, finally to a three dimensional network, effects a greater resistance to degradation resp. decomposition induced by cells and the cell culture medium. Most degradation, so is reported, causes lipid absorption and altering the nature of filler–binder interactions play a major role too [20,21]. In addition, the initially very weak material gains strength due to the electron beam treatment. As in original silicones (e.g. tubes) are particularly soft materials, their mechanical properties can be significantly improved by customized electron beam treatment. For
each area of application of silicone materials, optimum treatment parameters for the material properties must be determined. The contact angle, surface energy, and number of polar surface groups are key parameters when discussing wetting phenomena, because when two phases come into contact with each other only similar groups interact. The increase in the polar groups is caused by reactions of oxygen with polymer molecule radicals which simultaneously result in the lowering of the contact angle [22]. The oxygen content on the surface arises very fast just at the beginning of the treatment [14]. Comparative studies by the authors using plasma treatment and electron beam treatment resulted in a much greater increase in the surface energy for plasma treatment, and in particular of the polar contributions (Fig. 10), [16,23,24]. Analogously, the cell adhesion tests showed a factor of 4 higher number of fibroblasts. In general it was established that the fibroblasts proliferate to a greater extent on surfaces having a low contact angle (Fig. 11). Other literature also reports that cell adhesion is higher on hydrophilic, energy-rich surfaces [22,24,25]. This trend could only be confirmed to a limited extent via the electron beam treatment. Basically, it was found that the adhesion of the fibroblasts drastically decreased even though the hydrophilicity and the surface energy slightly increased. This phenomenon has also been observed by other authors [26,27] and not only indicates the complexity of the treatment, but also opens the door for many potential applications. There is still no absolute clarity about the exact mechanism of cell adhesion. There is agreement, however, that not only the surface energy, roughness, texture and charge affect cell adhesion, but also chemical reactions of the surface groups, crystallinity and mechanical properties which can promote or hinder the adhesion [28–31]. The magnitude of such effects of individual parameters on the adhesion and which role the combination of specific parameters does play still must be determined. There are applications of the experimental results the authors see in the therapeutically medical technics with polymer products. The functionality can be perfectly adapted for cell adherents and also for chemical and physical properties with “surface-gradient-layers”. 5. Conclusions These studies on polyurethane and silicone have shown that electron beam treatment is a suitable tool for modifying polymers. The electron beam treatment of polyurethane resulted in lower adhesion of fibroblasts (L929). No fibroblasts adhered to silicone — either untreated or electron beam treated. However, the resulted modification of the surface molecules appears to protect the material against damage by the cells and their enzymes. The higher stiffness caused by the electron beam treatment – for equivalent implant geometry – is however very important for adapting medical products and could provide an improved “stress shielding” effect. The application-specific optimization of the biofunctionality and the simultaneous sterilizing effect make the electron beam treatment a very promising method for a wide range of uses. The aim of ongoing further work is the identification of parameters for specific applications, so opening up a number of applications in therapeutic medical technics. The authors thank Steffen Strehle (University of Technology, Dresden) and Konrad Schneider (Leibniz Institute of Polymer Research, Dresden) for their assistance in XPS and mechanical tests. References
Fig. 11. Proliferation rate as a function of the contact angle of polyurethane.
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