Surface Modification of the Polytetrafluoroethylene Films with Treatment of Low Energy Ion Beams

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1 Biomaterials Research (006) 10(4) : Biomaterials Research 7 The Korean Society for Biomaterials Surface Modification of the Polytetrafluoroethylene Films with Treatment of Low Energy Ion Beams Yoon Jeong Choi, Mi-Sook Kim, and Insup Noh* Department of Chemical Engineering, Seoul National University of Technology, Seoul , Korea (Received October 13, 006/Accepted November 3, 006) Even though surface modification of polytetrafluoroethylene (PTFE) has led to improvement of its cellular interaction, its applications in tissue engineering for vascular graft patency are less than its initial hopes. To increase initial smooth muscle cell adhesion, its surface modification of PTFE film and its subsequent evaluation were performed after predetermining the irradiation conditions with Stopping and Range of Ions in Matter software by irradiating argon ion beams with low energy on the surfaces of PTFE. We here studied a possibility of surface modification of PTFE by employing irradiation of kev argon ion beams at a density of ions/cm. The extent of surface modification was evaluated by observing its color and morphological changes with digital image camera and scanning electron microscopy, respectively, as well as measuring chemical composition changes with X-ray photoelectron microscopy. Furthermore in vitro cell culture was performed on the argon ion beam-treated surfaces, and their cellular adhesions were evaluated with cell counting kit by detecting optical density of the medium placing in the cellcultured sample with microplate-reader. Cellular interaction of the argon ion beam treated surface induced increases of cell adhesion on the surfaces of the beam treated PTFE film, demonstrating that argon beam-treated surface induced higher cell adhesion than the untreated one did. Key words: Ion beam irradiation, Surface modification, Polytetrafluroethylene, Films, Smooth muscle cell adhesion C INTRODUCTION *Corresponding author: insup@snut.ac.kr ardiovascular disease has been known to be one of the leading causes of the death in the developed countries and more than 570,000 artery bypass graft surgeries are performed each year in the United States, percutaneous devices have still abounded in extreme cases. 1) Large caliber expanded polytetrafluoroethylene (PTFE) (>5 mm) are clinically employed with reasonable results, but its clinical applications in smaller caliber vessels is still problematic.,3) High patency grafts has been achieved to some extent achieved by numerous methods of PTFE, but its results are less than its initial hopes. Surface modification has been employed on the vascular scaffolds such as PTFE, Dacron and other polymers. As examples, surface modifications of PTFE film with either argon ion beam or UV light from Xe-excimer lamp were reported to increase its interaction with vascular endothelial cell targeting to the improvement of small caliber PTFE patency. 4) Another examples of surface modifications and its evaluations have been tried such as carbon nitride (CN x ) ions deposited on PTFE films by using the ion beam-assisted deposition technique, 5) chemical treatmet with anthraquinones and enzophenone 3,6) and plasma treatment, 7,8) and further evaluations of bioactive molecule transport through the graft wall for tissue engineering applications. 9) New method of surface modification of PTFE films by irradiation of argon ion beams with low energy is being here reported as a preliminary study to solve our previously reported tissue engineering-related issues of the hybrid PTFE scaffold, where consisted of PTFE and porous biodegradable poly(lactide-co-glycolide) layers on its surfaces. 3,9-11) Regeneration of media tissues on its biodegradable poly(lactide-co-glycolide) layer was reported by in vitro culturing porcine vascular smooth muscle cells. 9-11) Further study on a second step surface modification of expanded PTFE, 1-14) its induction of new tissue regeneration, transport characteristics and stability of the regenerated tissue were subsequently reported under circulation of pulsatile flow. 9) Its regenerated tissue demonstrated under very harsh in vivo recirculation conditions good performances such good mechanical stability and permeability through its wall. As a way of further enhancing cellular interaction on the PTFE surface of the hybrid scaffold after tissue replacement of the biodegradable layers with proliferating smooth muscle cells, we here surface-modified PTFE by treating with argon ion beams with low energy, and evaluated in visual, morphological and chemical the extent of its modifications as well following its in vitro cell adhesion. 01

2 0 Yoon Jeong Choi, Mi-Sook Kim, and Insup Noh MATERIALS AND METHODS Surface Modification of Polytetrafluoroethylene Films PTFE Film Preparation We prepared PTFE films for observation of surface modification and cell interaction behaviors. First, the PTFE films (Jinil Tec-PLA Inc., Korea) were cut into a round shape, having a dimension of 1 cm diameter and 0.4 mm thickness. PTFE films were dried after their extraction with dichloromethane for overnight. Ion Beam Treatment on PTFE Film Surface Treatment of the above PTFE films was performed by irradiating on their sample surfaces with argon ion beams with low energy ( kev, ions/cm ) by Dual Ion Beam Implantation System (Korea Atomic Energy Research Institute, Daejeon, Korea). After loading the dry samples on the sample stage, the pressure in the stage was reduced to in vacuuo for ion beam irradiation and then argon gas from the ion source was fed to the sample surface. The rates of the argon ion beams were accelerated by controlling electrical potential of the acceleration tube, generating acceleration of ion beam energy from 1~ kev for approximately 30 min to 1 hr depending upon the sample treatment conditions. The actual conditions of the argon ion beams such as ion energy and intensity were in advance determined by utilizing Stopping and Range of Ions in Matter (SRIM) software, i.e. by informing data about polymer species and density of the samples as well as its glass transition temperature. Characterization of the Ion Beam-irradiated PTFE Films Morphological Changes of the Ion Beam-irradiated PTFE Films Overall images of the the ion beam-irradiated PTFE samples such as morphologies and colors were captured with a digital camera (Cannon Inc., Japan). For morphological images with scanning electron microscopy, sputter coating of the samples was performed with gold in plasma for 60 sec, thus forming 00Å coated-thickness. After inserting the coated PTFE samples into a vacuum chamber of SEM (JSM-6400; Joel Ltd, Japan), morphological images of the films and their cell-cultured samples were obtained from 10- to 3,000-times magnifications to observe the existence of morphological changes and cell adhesion on their surfaces. Chemical Composition Changes with X-ray Photoelectron Spectroscopy (XPS) We observed chemical composition changes of the ion beam-irradiated PTFE film surfaces with a monochromator Al- Ká spectrometer XPS (PHI 5800 ESCA system; U.S.A.). After obtaining the XPS spectra of the survey scans in the range of 0-1,400 ev, C1s high resolution spectra were obtained after either referencing binding energy of the fluorocarbon at 9.5 ev for the untreated films or at 84.5 ev for the beam-irradiated films. Characterization of Cellular Interaction of the Ion Beam-irradiated PTFE Films Porcine arterial smooth muscle cells under passage 7 were in vitro cultured on the PTFE surfaces (n=3). After sterilizing the samples, in vitro cell culture was performed on their surfaces at densities of either 10,000 cells/cm, 50,000 cells/cm or 00,000 cells/cm for 1 hr in DMEM growth media containing 10% fetal bovine serum and penicillin-streptomycin (100 IU/ml-100 µg/ml, respectively) at 37 o C and 5% CO under static conditions in an incubator. For an observation of the cell adhesion morphologies, the cell-cultured samples were rinsed with phosphate buffered solution (PBS) solution to remove non-adherent cells, and then fixed with 3% formaldehyde in PBS solution. Following rinsing the samples three times with PBS solution, the samples were consecutively dried with graded ethanols of 70, 80, 90, 95, and 100%. Morphologies of the samples were observed with SEM for observations of adherent cells as described above. The number of cell adhesion on the cell-cultured samples were evaluated with cell counting kit (CCK-8, Japan) by detecting optical density of the medium containing the cell-cultured sample at a wavelength of 450 nm with Microplate-reader (Tecan, Austria). Figure 1. Morphology and color changes of the PTFE films: the surface morphologies before [(A) by digital picture and the left side one of (B) by SEM] and after beam treatment [(C) by digital picture and the right side one of (B) by SEM]. Biomaterials Research 006

3 Surface Modification of the Polytetrafluoroethylene Films with Treatment of Low Energy Ion Beams 03 RESULTS Treatments of the PTFE film surfaces were performed with irradiation of argon ion beams with the energy of kev, respectively. Its surface modification was clearly obtained by observing visually the color changes of the PTFE film from white to brown, but its overall morphologies seemed to be preserved as observed by digital images (Figure 1-B). Further observation of for the PTFE film was performed by magnifying surface morphologies with SEM. The SEM images of the argon beam treated surfaces (Figure 1-C) demonstrated no significant morphological changes compared those of the untreated ones (Figure 1-A). Surface chemical structure changes of the ion beam-irradiated PTFE samples were also measured by XPS by referencing the untreated PTFE with chemical structure of [ (CF -CF -) n -]. On their survey scans, the untreated PTFE sample contained its chemical composition of carbon (36%) and fluorine (63%) with minor oxygen atoms (Table 1), indicating that the polymer sample was polytetrafluoroethylene with a fluorine to carbon chemical composition ratio of about :1. Treatment of the PTFE films with argon ion beam led to incorporation of oxygen atoms as clearly obtained as 10% from the PTFE films, with 4% nitrogen atomic composition incorporated, as shown in both Figure and Table 1. The oxygen atomic was clearly qualitatively incorporated in the film surface as shown in Figure, which was expected to be incorporated into some depth of its surfaces. To understand defluorination and following incorporation of oxygen atoms into the sample surface, we analyzed in detail its high resolution C1s peak. Defluorination and subsequent incorporation of oxygen atoms were clearly observed in the C1s high resolution of the treated PTFE film by peak changes such as decrease of its original carbon peak at 9.5 ev (Figure 3). This surface modification generated new sharp carbon peaks at lower binding energy, i.e. exactly 84.5 ev (Figure 3-B), even though its original fluorine peak was observed in its survey scan (Figure ), while the untreated PTFE samples demonstrated its typical minor, remnant fluorocarbon peak at 9.5 ev (Figure 3-A). We assumed that defluorination was induced by the effect of incorporation of the accelerated argon ion beams into certain depth of PTFE wall thickness, approximately 7 nm, under this kev ion beam energy irradiation. The existence of oxygen atoms on of the argon ion beam-irradiated PTFE films as shown in both the survey scan and the C1s high resolution seemed to be by the exposure of the argon ion beam-treated sample to air environment right after its beam treatment, which might have various reactive species such as radicals, ions and atoms. This new carbon peak seemed to be matched with the results of its color changes to pale to dark black color, where the unsaturated carbon peaks have been known to be in black color and to have their hydrocarbon unsaturated carbon binding Table 1. Chemical compositions of the PTFE films by XPS survey scan. Chemical composition Films C 1S (%) N 1S (%) O 1S (%) F 1S (%) Untreated PTFE Argon-treated PTFE Figure. Survey scans of the PTFE film surface by XPS; the untreated PTFE (A) and the argon beam treated PTFE (B). Figure 3. C 1S high resolutions of the PTFE surfaces by XPS; the untreated PTFE (A) and the argon beam treated PTFE (B). energy at around 84.5 ev. We further examined the effect of argon ion beam treatment on the adhesion behaviors of in vitro smooth muscle cell on the PTFE surfaces with SEM. The PTFE film induced in vitro cell adhesion by the effect of argon ion beams on the PTFE film surface, when in vitro smooth muscle cell culture was performed for 1 hr. While the untreated PTFE surface demonstrated nearly no cell adhesion (Figures 4-A, -B and -C), the argon ion beam treated surface clearly induced in vitro Vol. 10, No. 4

4 04 Yoon Jeong Choi, Mi-Sook Kim, and Insup Noh Smooth muscle cell adhesion on the PTFE surface cultured for 1hr dependent on cell loading density of 10,000 cells/cm (A and D), 50,000 cells/cm (B and E) and 00,000 cells/cm (C and F); (A), (B) and (C) for the untreated PTFE and (D), (E) and (F) for the argon-beam treated PTFE. Figure 4. cell adhesion (Figures 4-D, -E and -F). As we increase the number of cells on the film surface when cell-cultured, the degree of cell adhesion was increased. In specific, higher cell adhesion was observed on both surfaces treated with either 50,000 or 00,000 cells/cm, than that with 10,000 cells/cm. After observing cell adhesion on, we further measured how many cells were adhered on the ion beam-irradiated PTFE surfaces by employing microplate reader dependent on the cell loading density (Figure 5). We could observe significant differences in the number of cells adhered: The beam-treated PTFE surfaces induced always higher number of cells adhered than the untreated surfaces did. Remarkably increased cell adhesion was observed on the beam-treated surfaces when the highest number of cells was cultured such as 00,000 cells/cm. Measurement of smooth muscle cell adhesion on the surfaces of the PTFE films after cell culture at different cell loading density. Figure 5. Biomaterials Research 006

5 Surface Modification of the Polytetrafluoroethylene Films with Treatment of Low Energy Ion Beams 05 DISCUSSION AND CONCLUSION Surface modifications of the PTFE vascular graft surface were successfully obtained by irradiating low energy argon ion beams as observed by color and chemical composition changes without changes in their surface morphologies. When we measured the extents of surface modification on the chemical composition by XPS as well as the depth of ion beam penetration from surface, chemical modification seemed to be performed by observing new chemical species such as unsaturations and oxygen atoms. The extent of argon beam penetration was calculated to be to depth of 7 nm for the employment of kev ion beams in this study. This surface modification by argon ion beam irradiation on the PTFE films induced signification increase in the number of cell adhesion as observed by SEM, qualitatively, and cell counting kit measurement, quantitatively. Clear cell adhesion and limited cell spreading were observed on the SEM pictures. We expect this new method of surface modification lead to increase in interaction between cells, regenerated tissues and modified eptfe surface, which will be core components of our previous hybrid eptfe vascular scaffolds. 3,9-13) ACKNOWLEDGEMENT This study was supported by a grant of the Korea Health 1 R&D Project, Ministry of Health & Welfare, Republic of Korea (A05008). REREFENCES 1. T. Neumann, B. S. Nicholson, and J. E. Sanders, Tissue engineering of perfused microvessels, Microvasc. Res., 66, (003).. T. Shinoka, D. Shum-Tim, P. X. Ma, R. E. Tanel, N. Isogai, R. Langer, J. P. Vacanti, and J. E. Jr. Mayer, Creation of viable pulmonary artery autografts through tissue engineering, J. Thorac. Cardiovasc. Surg., 115, (1998). 3. I. Noh and E. R. Edelman, Smooth muscle cell ingrowth of a surface-modified eptfe vascular graft, Key Eng. Mat., 88-89, (005). 4. R. Mikulikova, S. Moritz, T. Gumpenberger, M. Olbrich, C. Romanin, L. Bacakova, V. Svorcik, and J. Heitz, Cell microarrays on photochemically modified polytetrafluoroethylene, Biomaterials, 6, (005). 5. X. L. Qing, C. L. Zheng, and F. Z. Cui, In vitro endothelialization on CNx films deposited on PTFE, J. Korean Phys. Soc., 46, 4-9 (005). 6. J. S. Lee, B. Y. Kim, and J. H. Lee, A study on the change in the microstructure and the surface properties of polymers by ion implantation, J. Korean Phys. Soc., 47, (005). 7. M. A. Golub, Concerning apparent similarity of structures of fluoropolymer surfaces exposed to an argon plasma or argon ion beam, Langmuir, 1, (1996). 8. K. P. Grytsenko and S. Schrader, Nanoclusters in polymer matrices prepared by co-deposition from a gas phase, Adv. Colloid Interface Sci., 116, (005). 9. Y. J. Choi, S. K. Choung, C. M. Hong, I. S. Shin, S. N. Park, S. H. Hong, H. K. Park, Y. H. Park, Y. Son, and Noh I., Evaluations of blood compatibility via protein adsorption treatment of the vascular scaffold surfaces fabricated with polylactide and surfacemodified expanded polytetrafluoroethylene for tissue engineering applications, J. Biomed. Mater. Res., 75, (005). 10. I. Noh, Vascular tissue regeneration of the hybrid eptfe graft for adult patients, Key Eng. Mat., 88-89, (005). 11. I. Noh and J. A. Hubbell, Photograft polymerization of acrylate monomers and macromonomers on photochemically reduced PTFE films, J. Polym. Sci. Part A: Polym. Chem., 35, (1997). 1. Y. J. Choi and I. Noh, Surface modification of the polytetrafluoroethylene film with cyclotron ion beams and its evaluation for applications in tissue engineering, Surf. Coat. Technol. (006, on-line). 13. J. H. Noh, H. K. Baik, I. Noh, J. C. Park, and I. S. Lee, Surface modification of polytetrafluoroethylene using atmospheric pressure plasma jet for medical application, Surf. Coat. Technol. (006, on-line). 14. Y. J. Choi, S. H. Moon, M. S. Kim, and I. Noh Evaluation of a porous gelatin-eptfe scaffold for its potential applications in vascular grafts, Biomater. Res., 10, (006). Vol. 10, No. 4