- Email: [email protected]
Ion beam modification of ePTFE for improving the blood compatibility
Surface & Coatings Technology 206 (2011) 905–910
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
Ion beam modification of ePTFE for improving the blood compatibility Hitomi Hiruma a,b,⁎, Hiroshi Toida b, Takao Hanawa a, Hitoshi Sakuragi b, Yoshiaki Suzuki b a b
Department of Metals, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Artificial Organ Materials Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2–1 Hirosawa, Wako-shi, Saitama 351–0198, Japan
a r t i c l e
i n f o
Available online 2 April 2011 Keywords: Expanded polytetrafluoroethylene (ePTFE) Surface modification Ion beam Anti-thrombogenicity Platelet adhesion Endothelial cell adhesion
a b s t r a c t Expanded polytetrafluoroethylene (ePTFE), which is a durable biomaterial because of its excellent biological inertness, is now widely used for prostheses in clinical medicine. However, conversely, the inert nature of ePTFE results in poor adaptability to the surrounding tissue due to lack of a cell-adhesive property. In this study, the surface of ePTFE was modified with ion beam irradiation to improve the blood compatibility. The surface modification of ePTFE sheets by He+, Ne+, Ar+ and Kr+ ion beams was performed at an energy of 150 keV with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2. To investigate anti-thrombogenicity, Ca2+replenished platelet-rich plasma (PRP) was placed in contact with the surfaces for 10 min. Compared to the non-modified ePTFE surface, platelet response was inhibited on the surfaces modified with He+, Ne+ and Ar+: 5 × 1014 and 1 × 1015 ions/cm2, and Kr+: 5 × 1014 ions/cm2; however, platelet response was promoted on the surfaces modified with He+, Ne+ and Ar+: 1 × 1014 ions/cm2, and Kr+: 1 × 1014 and 1 × 1015 ions/cm2. The significant morphological changes in ePTFE surface associated with ion beam modification are thought to be one of the reasons for the inhibition of platelet response. Endothelial cells were cultured on the surfaces for 3 days to evaluate the cellular response. Endothelial cell growth was significantly promoted on all of the surfaces of ion beam-modified ePTFE, although the non-modified ePTFE surface dramatically inhibited this growth. It is concluded that ion beam modification of ePTFE surface can improve the blood compatibility through not only the promotion of endothelial cell growth but also the inhibition of platelet response. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Expanded polytetrafluoroethylene (ePTFE) is a chemically stable polymer and a durable biomaterial because of its excellent biological inertness. In clinical medicine, ePTFE is now widely used for prostheses such as vascular grafts [1], dural substitutes [2], pericardial substitutes [3], peritoneal patches [4], artificial tendinous cords [5], barrier membranes for guided tissue regeneration [6], suture materials [7], and so on. However, conversely, the characteristic advantages of ePTFE such as inactivity result in poor adaptability to the surrounding tissue due to lack of a cell-adhesive property, and also a surgical tissue adhesive such as applied fibrin glue is repelled. Recently, our previous studies reported that surface modification using ion beam irradiation could change the characteristics of the surface of ePTFE to enable it to adhere to the surrounding tissue and fibrin glue [8]. Therefore, ion beam-modified ePTFE demonstrates great sealing and is ideal for an artificial dura mater [9,10] and an aneurysm wrapping material [11]. Furthermore, the blood compati-
⁎ Corresponding author at: Department of Metals, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel./fax: + 81 3 5280 8009. E-mail address: [email protected] (H. Hiruma). 0257-8972/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2011.03.134
bility of ePTFE is a very important factor for its use for bloodcontacting materials such as graft materials for artificial vascular grafts and stent grafts, cover materials for covered stents, reparative materials for small injuries to vessel walls and organs, supporting materials for vascular anastomosis in micro-surgery and vascular surgery, and so on. In this study, the surface of ePTFE was modified by using ion beam irradiation to produce both an anti-thrombogenic surface and an endothelial cell-adhesive surface. The anti-thrombogenicity and endothelial cell-adhesive property of ePTFE surface were evaluated by means of in vitro platelet adhesion and endothelial cell adhesion experiments, respectively. 2. Materials and methods 2.1. Specimen and ion beam surface modification ePTFE sheets (PSM-01200, W. L. Gore and Associates, USA) of which the size was 100 μm in thickness, were cut into 10 mm × 10 mm squares, and were used as specimens for this study. The surface modification of the ePTFE sheets utilizing He+, Ne+, Ar+ and Kr+ ion beams was carried out at an energy of 150 keV with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2. The current density of the ion beams was 0.05 μA/cm2. The pressure of the target chamber during ion beam irradiation was maintained at a base value of 10− 4 Pa.
906
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
2.2. Platelet studies
3. Results
Blood was withdrawn from a healthy female, using a sterile 21-G needle. Coagulation of the blood was inhibited with tri-sodium citrate (3.8% w/v in H2O) at a volume ratio of 1 part citrate to 9 parts blood. Whole blood was immediately centrifuged at 110 G for 15 min to prepare platelet-rich plasma (PRP). After collecting the supernatant, the remainder was centrifuged further at 1120 G for 15 min to prepare platelet-poor plasma (PPP). Then, PRP was adjusted to a concentration of 1 × 105 platelets/μl using PPP. For the platelet activation experiments, 85 μl of 0.25 M CaCl2 was added to 1 ml PRP, and 50 μl PRP was dropped on the surfaces of the specimens and then incubated at 37 °C. After 10 min, the specimens were rinsed with PBS 2 times to remove non-adhered/loosely bound platelets and then fixed in 2.5% (v/v) glutaraldehyde overnight and dehydrated in 30% (v/v), 50% (v/v), 70% (v/v), 90% (v/v) and 100% (v/v) ethanol for 15 min for each concentration. The specimens were dried at room temperature and sputter-coated with gold, and then observed using SEM (JSM6330F, JEOL, Japan). The SEM images of at least 3 different regions were analyzed.
3.1. Platelet response
Bovine aortic endothelial cells (BAECs), isolated from the descending aorta by treatment with 0.1% collagenase, were used. BAECs were cultivated on a tissue culture polystyrene dish in RPMI 1640 (Nissui Pharmaceutical Company, Japan) supplemented with 10% fetal bovine serum (CCT, Sanko Junyaku Co., Ltd., Japan), 5.7 mg/ml penicillin, 10 mg/ml streptomycin and 2.5 μg/ml amphotericin, at 37 °C in a humidified atmosphere of 5% CO2. BAECs were subcultured at subconfluences and used between the seventh and the ninth passage.
Platelet adhesion, activation and aggregation are the indications of thrombus formation on a surface. Fig. 1 shows the SEM images of platelets adhered onto a non-modified ePTFE surface as a control and onto the ion beam-modified ePTFE surfaces after contact with Ca2+-replenished PRP for 10 min. The total number of platelets adhered onto these surfaces was counted from the SEM images, as shown in Fig. 2. These data are presented as mean ± standard deviation. Platelet activation on all of the surfaces of ion beam-modified ePTFE was almost the same as that on the non-modified ePTFE surface, although the total number of adhered platelets showed statistically significant differences between these. Compared to the number of platelets adhered onto the non-modified ePTFE surface, the number of adhered platelets decreased on the surfaces modified with He+, Ne+ and Ar+: 5 × 1014 and 1 × 1015 ions/cm2, and Kr+: 5 × 1014 ions/cm2; however, the number of adhered platelets increased on the surfaces modified with He+, Ne+ and Ar+: 1 × 1014 ions/cm2, and Kr+: 1 × 1014 and 1 × 1015 ions/cm2. The number of platelets adhered onto He+ and Ne+ ion beam-modified surfaces decreased as the fluence increased. In the case of Ar+ and Kr+ ion beam-modified surfaces, the number of adhered platelets decreased up to a fluence of 5 × 1014 ions/cm2, and increased at a fluence of 1 × 1015 ions/cm2. Focusing on fluence, the number of adhered platelets at a fluence of 1 × 1014 ions/cm2 was higher than that for the non-modified ePTFE in all of the ion species, and increased as the mass number of the ion species increased. For the fluences of 5 × 1014 and 1 × 1015 ions/cm2, the number of platelets adhered onto Ar+ and Ne+ ion beam-modified surfaces showed the minimum value between other ion species, respectively.
2.4. Endothelial cell adhesion test
3.2. Endothelial cell growth
For the endothelial cell adhesion studies, BAECs were seeded onto a non-modified ePTFE surface and onto each of the ion beam-modified ePTFE surfaces at a density of 3 × 104 cells/dish and cultured for 3 days. After that BAECs were rinsed with PBS 2 times to remove nonadhered/loosely bound cells and then fixed in 2.5% (v/v) glutaraldehyde overnight and dehydrated in 30% (v/v), 50% (v/v), 70% (v/v), 90% (v/v) and 100% (v/v) ethanol for 15 min in each concentration. The specimens were dipped in 100% ethanol for observation by phasecontrast microscopy (IX70, OLYMPUS, Japan). The phase-contrast micrographs of at least 3 different regions were analyzed. In order to determine the number of adhered cells, BAECs were washed with PBS and trypsinized with 0.25% Trypsin-EDTA (GIBCO, USA) and then counted with a hemocytometer. As a positive control, tissue culturetreated polystyrene (TCPS 24-Multiwell Plates; AGC TECHNO GLASS CO., LTD., Japan) was also evaluated.
Endothelial cell adhesion, spreading and proliferation have the potential to enhance endothelium lining (i.e. endothelialization) on a surface. Fig. 3 shows the phase-contrast micrographs of BAECs adhered onto a non-modified ePTFE surface as a control and onto the ion beam-modified ePTFE surfaces after incubation for 3 days. The total number of BAECs adhered onto these surfaces is shown in Fig. 4. These data are presented as mean ± standard deviation. Tissue culture-treated polystyrene (TCPS) also is shown, as a positive control. Endothelial cell adhesion, spreading and proliferation were observed on all of the surfaces of ion beam-modified ePTFE. In contrast, endothelial cell attachment was dramatically inhibited on the non-modified ePTFE surface, and it was virtually impossible to grow the cells on this surface. The number of adhered endothelial cells dramatically increased on all of the surfaces of ion beam-modified ePTFE, compared to that on the non-modified ePTFE surface. For the surfaces modified with Ne+ and Kr+: 5 × 1014 ions/cm2, the number of adhered endothelial cells was higher than that on the TCPS used as a positive control. The number of endothelial cells adhered onto He+, Ne+ and Kr+ ion beam-modified surfaces increased up to a fluence of 5 × 1014 ions/cm2, and decreased at a fluence of 1 × 1015 ions/cm2. In the case of Ar+ ion beam-modified surfaces, the number of adhered endothelial cells increased as the fluence increased. Focusing on fluence, the number of adhered endothelial cells at a fluence of 1 × 1014 ions/cm2 showed no significant difference between the ion species. For a fluence of 5 × 1014 ions/cm2, the number of endothelial cells adhered onto Ar+ ion beam-modified surface showed the minimum value between other ion species. The number of adhered endothelial cells at a fluence of 1 × 1015 ions/cm2 slightly decreased as the mass number of the ion species increased.
2.3. Endothelial cell culture
2.5. Observation and analysis of surface morphology The surface morphology of the specimens was observed by means of SEM (JSM6330F, JEOL, Japan) after sputter-coating with gold in a plasma coater (SC-701, Sanyu Denshi Co., Ltd., Japan). The average crack size in the surfaces of the specimens was estimated with ImageJ software [12]. First, we converted the SEM image of the surface morphology of a specimen to an image segmented into black and white using a threshold function, and a median filter was applied to minimize the noise by calculating the average grayscale in a pixel from the values in the neighboring pixels. Then, the particle size corresponding to the crack in the surface was estimated with the ‘Analyze particles’ features of this software.
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
907
Control 10µm
He+
Ne+
Ar+
Kr+
1x1014 ions/cm2
5x1014 ions/cm2
1x1015 ions/cm2
Fig. 1. SEM images of human platelets adhered onto the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV, after contact with Ca2+-replenished PRP for 10 min.
3.3. Change in surface morphology The surfaces of non-modified and ion beam-modified ePTFE were observed by using SEM to examine the morphological changes in the ePTFE surface associated with ion beam modification. The nonmodified ePTFE had a micro-porous structure consisting of the submicron-sized nodes and fibrils of PTFE. After ion beam modification of the ePTFE surface, the node size decreased and the fibrils disappeared near the surface. Consequently, the pore size increased near the surface [9,11]. As a result, many cracks among the nodes were formed in ion beam-modified ePTFE surfaces, as seen in Fig. 1. In order to measure the average crack size in the ePTFE surfaces, the SEM images of the ePTFE surfaces were analyzed by using ImageJ software. The ImageJ software allows us to count the number of black pixels in each crack for every horizontal line of an image. However, many small pixels are in the SEM images of the ePTFE surfaces, which
have very fine structure, and therefore the average crack size in the ePTFE surfaces is estimated to be small. Fig. 5 shows the average crack size in a non-modified ePTFE surface as a control and in the ion beammodified ePTFE surfaces. After ion beam modification of the ePTFE surface, the average crack size increased in all of the surfaces of ion beam-modified ePTFE, compared to that in the non-modified ePTFE surface. The average crack size in He+, Ne+ and Ar+ ion beam-modified surfaces increased as the fluence increased. In the case of Kr+ ion beam-modified surfaces, the average crack size increased up to a fluence of 5 × 1014 ions/cm2, and decreased at a fluence of 1 × 1015 ions/cm2. Focusing on fluence, the average crack size at a fluence of 1 × 1014 ions/cm2 slightly increased as the mass number of the ion species increased. For the fluences of 5 × 1014 and 1 × 1015 ions/cm2, the average crack size in Ne+ ion beam-modified surfaces showed the maximum value between other ion species.
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
Number of adhered platelets [platelets/mm2]
908
4. Discussion
35 1×1014 ions/cm2
30
5×1014 ions/cm2
4.1. Platelet adhesion
1×1015 ions/cm2
25 20 15 10 5 0
Control
He+
Ne+
Ar+
Kr+
Fig. 2. Total number of platelets adhered onto the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV. These data are presented as mean ± standard deviation.
The foreign surfaces of artificial biomaterials for medical devices cause thrombus formation, which remains as one of the major problems in the development of blood-contacting materials. The thrombus formation on the surface is closely related to the surface properties such as surface energy, surface potential, surface morphology, surface chemical structure such as functional groups and characteristic groups, wettability (i.e. hydrophilicity and hydrophobicity) on the surface, and so on. Ion beam surface modification of ePTFE caused morphological changes in the surface, compared to the non-modified ePTFE surface. Comparing between Figs. 5 and 2, one can observe correlations between the average crack size and the total number of adhered platelets for ion beam-modified ePTFE surfaces. That is, the enlargement of the average crack size due to increasing the fluence for each ion species resulted in a decrease in the number of adhered platelets, and conversely, the reduction of the average crack size due to
Fig. 3. Morphology of BAECs cultured for 3 days on TCPS and the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV.
Number of adhered cells [x104 cells/ml]
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
20 18
1×1014 ions/cm2
Initial cell number
5×1014 ions/cm2
16
1×1015 ions/cm2
14 12 10 8 6 4 2 0
TCPS Control
He+
Ne+
Ar+
Kr+
Fig. 4. BAEC growth on TCPS and the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV. BAECs were inoculated with 3 × 104 cells/dish and the growth was monitored by counting cells over the course of 3 days. These data are presented as mean ± standard deviation.
increasing the fluence from 5 × 1014 to 1 × 1015 ions/cm2 for Kr+ ion resulted in an increase in the number of adhered platelets. However, for a fluence of 1 × 1014 ions/cm2, the slight enlargement of the average crack size due to increasing the mass number of the ion species resulted in a remarkable increase in the number of adhered platelets. This result is thought to be due to the influence of the change in the chemical structure of the surface layer of ePTFE associated with ion beam modification [9,11]. For the lower fluences such as 1 × 1014 ions/cm2, the radiation effects due to ion bombardment become greater for the heavier ions, and therefore the change in the chemical structure of the surface layer of ePTFE becomes greater for Kr+ ion beam modification. Therefore, it is thought that one of the reasons for the inhibition of platelet adhesion on ion beam-modified ePTFE surfaces is the enlargement of the crack size and the reduction of the node size in these surfaces, because of which the scaffolds for platelet adhesion would diminish in these surfaces. The inhibition of platelet adhesion, activation and aggregation has the potential to improve anti-thrombogenicity on the surface.
the surface, compared to that on the non-modified ePTFE surface, as shown in Fig. 4. In comparison between Figs. 5 and 4, there was no correlation between the average crack size and the total number of adhered endothelial cells for ion beam-modified ePTFE surfaces. However, compared to the non-modified ePTFE surface, ion beam surface modification of ePTFE caused the significant morphological changes in the surface, and endothelial cell adhesion, spreading and proliferation were significantly promoted on the ion beam-modified ePTFE surfaces. Ion beam surface modification of ePTFE also caused a change in the chemical structure of the surface layer; specifically, \CF2\ bonds of ePTFE were degraded, and NC_O, NC_Cb and \CF3 bonds were newly produced in the surface layer [9,11]. Therefore, it is thought that the surface roughness, carbonyl groups and unsaturated bonds in the ePTFE surface associated with ion beam modification are effective in cell adhesion, and therefore ion beam-modified ePTFE surfaces would enhance the endothelium lining due to the improvement of endothelial cell adhesion, spreading and proliferation. 4.3. Surface morphology Ion beam surface modification of ePTFE caused an increase in the average crack size in the surface, compared to that in the non-modified ePTFE surface, as shown in Fig. 5. However, conversely, the average crack size at the fluences of 5 × 1014 and 1 × 1015 ions/cm2 decreased as the mass number of the bombarded-ion increased from Ne+ to Kr+ ion. Also, the average crack size in Kr+ ion beam-modified surfaces decreased as the fluence increased from 5 × 1014 to 1 × 1015 ions/cm2. The cross-sectional images of ion beam-modified ePTFE surfaces indicate that ion beam surface modification of ePTFE formed spinelike nodes perpendicular to the surface rather than cracks [9,11]. Therefore, it is thought that the ridges of the spine-like nodes in the surfaces of ion beam-modified ePTFE are etched by ion bombardment by which the radiation effects become greater for the heavier ions or the higher fluences, and therefore the node size increases and the crack size decreases in these surfaces. 5. Conclusion
4
The surface of ePTFE was modified with ion beam irradiation to improve the blood compatibility, which was evaluated by in vitro platelet adhesion and endothelial cell adhesion tests. The surface modification of ePTFE sheets utilizing He+, Ne+, Ar+ and Kr+ ion beams was performed at an energy of 150 keV with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2. Platelet response was inhibited on the surfaces modified with He+, Ne+ and Ar+: 5 × 1014 and 1 × 1015 ions/cm2, and Kr+: 5 × 1014 ions/cm2; however, platelet response was promoted on the surfaces modified with He+, Ne+ and Ar+: 1 × 1014 ions/cm2, and Kr+: 1 × 1014 and 1 × 1015 ions/cm2, compared to the non-modified ePTFE surface. Although the nonmodified ePTFE surface dramatically inhibited endothelial cell growth, this growth was significantly promoted on all of the surfaces of ion beam-modified ePTFE. These results suggest that the surfaces of ion beam-modified ePTFE have the potential to enhance the formation of neointima (i.e. pseudointima) by endothelium lining through not only the promotion of endothelial cell growth but also the inhibition of platelet response. The neointima formed by endothelium lining brings about anti-thrombogenicity on the surface.
2
References
4.2. Endothelial cell adhesion Ion beam surface modification of ePTFE caused the powerful promotion of endothelial cell adhesion, spreading and proliferation on
16 1×1014 ions/cm2
14 Average crack size [µm 2]
909
5×1014 ions/cm2 1×1015 ions/cm2
12 10 8 6
0
Control
He+
Ne+
Ar+
Kr+
Fig. 5. Average crack size in the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV.
[1] H. Matsumoto, T. Hasegawa, K. Fuse, M. Yamamoto, M. Saigusa, Surgery 74 (1973) 519. [2] S. Yamagata, K. Goto, Y. Oda, H. Kikuchi, Neurol. Med. Chir. Tokyo 33 (1993) 582. [3] G. Bhatnagar, S.E. Fremes, G.T. Christakis, B.S. Goldman, J. Card. Surg. 13 (1998) 190. [4] R.A. Monaghan, S. Meban, Can. J. Surg. 34 (1991) 502.
910
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
[5] Y. Kawahira, T. Yagihara, H. Uemura, T. Ishizaka, K. Yoshizumi, S. Kitamura, Eur. J. Cardiothorac. Surg. 15 (1999) 289. [6] J. Gottlow, S. Nyman, J. Lindhe, T. Karring, J. Wennström, J. Clin. Periodontol. 13 (1986) 604. [7] G. Setzen, E.F. Williams, Plast. Reconstr. Surg. 100 (1997) 1788. [8] N. Takahashi, H. Ujiie, Y. Suzuki, M. Iwaki, T. Hori, No Shinkei Geka 32 (2004) 339. [9] Y. Suzuki, M. Iwaki, S. Tani, G. Oohashi, M. Kamio, Nucl. Instrum. Methods Phys. Res. B 206 (2003) 538.
[10] N. Takahashi, Y. Suzuki, H. Ujiie, T. Hori, M. Iwaki, T. Yamada, Nucl. Instrum. Methods Phys. Res. B 242 (2006) 61. [11] N. Takahashi, Y. Suzuki, H. Ujiie, M. Iwaki, T. Hori, T. Yamada, Surf. Coat. Technol. 201 (2007) 8150. [12] U.S. National Institutes of Health, World Wide Web electronic publication, http:// rsbweb.nih.gov/ij/.
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
Ion beam modification of ePTFE for improving the blood compatibility Hitomi Hiruma a,b,⁎, Hiroshi Toida b, Takao Hanawa a, Hitoshi Sakuragi b, Yoshiaki Suzuki b a b
Department of Metals, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Artificial Organ Materials Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2–1 Hirosawa, Wako-shi, Saitama 351–0198, Japan
a r t i c l e
i n f o
Available online 2 April 2011 Keywords: Expanded polytetrafluoroethylene (ePTFE) Surface modification Ion beam Anti-thrombogenicity Platelet adhesion Endothelial cell adhesion
a b s t r a c t Expanded polytetrafluoroethylene (ePTFE), which is a durable biomaterial because of its excellent biological inertness, is now widely used for prostheses in clinical medicine. However, conversely, the inert nature of ePTFE results in poor adaptability to the surrounding tissue due to lack of a cell-adhesive property. In this study, the surface of ePTFE was modified with ion beam irradiation to improve the blood compatibility. The surface modification of ePTFE sheets by He+, Ne+, Ar+ and Kr+ ion beams was performed at an energy of 150 keV with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2. To investigate anti-thrombogenicity, Ca2+replenished platelet-rich plasma (PRP) was placed in contact with the surfaces for 10 min. Compared to the non-modified ePTFE surface, platelet response was inhibited on the surfaces modified with He+, Ne+ and Ar+: 5 × 1014 and 1 × 1015 ions/cm2, and Kr+: 5 × 1014 ions/cm2; however, platelet response was promoted on the surfaces modified with He+, Ne+ and Ar+: 1 × 1014 ions/cm2, and Kr+: 1 × 1014 and 1 × 1015 ions/cm2. The significant morphological changes in ePTFE surface associated with ion beam modification are thought to be one of the reasons for the inhibition of platelet response. Endothelial cells were cultured on the surfaces for 3 days to evaluate the cellular response. Endothelial cell growth was significantly promoted on all of the surfaces of ion beam-modified ePTFE, although the non-modified ePTFE surface dramatically inhibited this growth. It is concluded that ion beam modification of ePTFE surface can improve the blood compatibility through not only the promotion of endothelial cell growth but also the inhibition of platelet response. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Expanded polytetrafluoroethylene (ePTFE) is a chemically stable polymer and a durable biomaterial because of its excellent biological inertness. In clinical medicine, ePTFE is now widely used for prostheses such as vascular grafts [1], dural substitutes [2], pericardial substitutes [3], peritoneal patches [4], artificial tendinous cords [5], barrier membranes for guided tissue regeneration [6], suture materials [7], and so on. However, conversely, the characteristic advantages of ePTFE such as inactivity result in poor adaptability to the surrounding tissue due to lack of a cell-adhesive property, and also a surgical tissue adhesive such as applied fibrin glue is repelled. Recently, our previous studies reported that surface modification using ion beam irradiation could change the characteristics of the surface of ePTFE to enable it to adhere to the surrounding tissue and fibrin glue [8]. Therefore, ion beam-modified ePTFE demonstrates great sealing and is ideal for an artificial dura mater [9,10] and an aneurysm wrapping material [11]. Furthermore, the blood compati-
⁎ Corresponding author at: Department of Metals, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel./fax: + 81 3 5280 8009. E-mail address: [email protected] (H. Hiruma). 0257-8972/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2011.03.134
bility of ePTFE is a very important factor for its use for bloodcontacting materials such as graft materials for artificial vascular grafts and stent grafts, cover materials for covered stents, reparative materials for small injuries to vessel walls and organs, supporting materials for vascular anastomosis in micro-surgery and vascular surgery, and so on. In this study, the surface of ePTFE was modified by using ion beam irradiation to produce both an anti-thrombogenic surface and an endothelial cell-adhesive surface. The anti-thrombogenicity and endothelial cell-adhesive property of ePTFE surface were evaluated by means of in vitro platelet adhesion and endothelial cell adhesion experiments, respectively. 2. Materials and methods 2.1. Specimen and ion beam surface modification ePTFE sheets (PSM-01200, W. L. Gore and Associates, USA) of which the size was 100 μm in thickness, were cut into 10 mm × 10 mm squares, and were used as specimens for this study. The surface modification of the ePTFE sheets utilizing He+, Ne+, Ar+ and Kr+ ion beams was carried out at an energy of 150 keV with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2. The current density of the ion beams was 0.05 μA/cm2. The pressure of the target chamber during ion beam irradiation was maintained at a base value of 10− 4 Pa.
906
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
2.2. Platelet studies
3. Results
Blood was withdrawn from a healthy female, using a sterile 21-G needle. Coagulation of the blood was inhibited with tri-sodium citrate (3.8% w/v in H2O) at a volume ratio of 1 part citrate to 9 parts blood. Whole blood was immediately centrifuged at 110 G for 15 min to prepare platelet-rich plasma (PRP). After collecting the supernatant, the remainder was centrifuged further at 1120 G for 15 min to prepare platelet-poor plasma (PPP). Then, PRP was adjusted to a concentration of 1 × 105 platelets/μl using PPP. For the platelet activation experiments, 85 μl of 0.25 M CaCl2 was added to 1 ml PRP, and 50 μl PRP was dropped on the surfaces of the specimens and then incubated at 37 °C. After 10 min, the specimens were rinsed with PBS 2 times to remove non-adhered/loosely bound platelets and then fixed in 2.5% (v/v) glutaraldehyde overnight and dehydrated in 30% (v/v), 50% (v/v), 70% (v/v), 90% (v/v) and 100% (v/v) ethanol for 15 min for each concentration. The specimens were dried at room temperature and sputter-coated with gold, and then observed using SEM (JSM6330F, JEOL, Japan). The SEM images of at least 3 different regions were analyzed.
3.1. Platelet response
Bovine aortic endothelial cells (BAECs), isolated from the descending aorta by treatment with 0.1% collagenase, were used. BAECs were cultivated on a tissue culture polystyrene dish in RPMI 1640 (Nissui Pharmaceutical Company, Japan) supplemented with 10% fetal bovine serum (CCT, Sanko Junyaku Co., Ltd., Japan), 5.7 mg/ml penicillin, 10 mg/ml streptomycin and 2.5 μg/ml amphotericin, at 37 °C in a humidified atmosphere of 5% CO2. BAECs were subcultured at subconfluences and used between the seventh and the ninth passage.
Platelet adhesion, activation and aggregation are the indications of thrombus formation on a surface. Fig. 1 shows the SEM images of platelets adhered onto a non-modified ePTFE surface as a control and onto the ion beam-modified ePTFE surfaces after contact with Ca2+-replenished PRP for 10 min. The total number of platelets adhered onto these surfaces was counted from the SEM images, as shown in Fig. 2. These data are presented as mean ± standard deviation. Platelet activation on all of the surfaces of ion beam-modified ePTFE was almost the same as that on the non-modified ePTFE surface, although the total number of adhered platelets showed statistically significant differences between these. Compared to the number of platelets adhered onto the non-modified ePTFE surface, the number of adhered platelets decreased on the surfaces modified with He+, Ne+ and Ar+: 5 × 1014 and 1 × 1015 ions/cm2, and Kr+: 5 × 1014 ions/cm2; however, the number of adhered platelets increased on the surfaces modified with He+, Ne+ and Ar+: 1 × 1014 ions/cm2, and Kr+: 1 × 1014 and 1 × 1015 ions/cm2. The number of platelets adhered onto He+ and Ne+ ion beam-modified surfaces decreased as the fluence increased. In the case of Ar+ and Kr+ ion beam-modified surfaces, the number of adhered platelets decreased up to a fluence of 5 × 1014 ions/cm2, and increased at a fluence of 1 × 1015 ions/cm2. Focusing on fluence, the number of adhered platelets at a fluence of 1 × 1014 ions/cm2 was higher than that for the non-modified ePTFE in all of the ion species, and increased as the mass number of the ion species increased. For the fluences of 5 × 1014 and 1 × 1015 ions/cm2, the number of platelets adhered onto Ar+ and Ne+ ion beam-modified surfaces showed the minimum value between other ion species, respectively.
2.4. Endothelial cell adhesion test
3.2. Endothelial cell growth
For the endothelial cell adhesion studies, BAECs were seeded onto a non-modified ePTFE surface and onto each of the ion beam-modified ePTFE surfaces at a density of 3 × 104 cells/dish and cultured for 3 days. After that BAECs were rinsed with PBS 2 times to remove nonadhered/loosely bound cells and then fixed in 2.5% (v/v) glutaraldehyde overnight and dehydrated in 30% (v/v), 50% (v/v), 70% (v/v), 90% (v/v) and 100% (v/v) ethanol for 15 min in each concentration. The specimens were dipped in 100% ethanol for observation by phasecontrast microscopy (IX70, OLYMPUS, Japan). The phase-contrast micrographs of at least 3 different regions were analyzed. In order to determine the number of adhered cells, BAECs were washed with PBS and trypsinized with 0.25% Trypsin-EDTA (GIBCO, USA) and then counted with a hemocytometer. As a positive control, tissue culturetreated polystyrene (TCPS 24-Multiwell Plates; AGC TECHNO GLASS CO., LTD., Japan) was also evaluated.
Endothelial cell adhesion, spreading and proliferation have the potential to enhance endothelium lining (i.e. endothelialization) on a surface. Fig. 3 shows the phase-contrast micrographs of BAECs adhered onto a non-modified ePTFE surface as a control and onto the ion beam-modified ePTFE surfaces after incubation for 3 days. The total number of BAECs adhered onto these surfaces is shown in Fig. 4. These data are presented as mean ± standard deviation. Tissue culture-treated polystyrene (TCPS) also is shown, as a positive control. Endothelial cell adhesion, spreading and proliferation were observed on all of the surfaces of ion beam-modified ePTFE. In contrast, endothelial cell attachment was dramatically inhibited on the non-modified ePTFE surface, and it was virtually impossible to grow the cells on this surface. The number of adhered endothelial cells dramatically increased on all of the surfaces of ion beam-modified ePTFE, compared to that on the non-modified ePTFE surface. For the surfaces modified with Ne+ and Kr+: 5 × 1014 ions/cm2, the number of adhered endothelial cells was higher than that on the TCPS used as a positive control. The number of endothelial cells adhered onto He+, Ne+ and Kr+ ion beam-modified surfaces increased up to a fluence of 5 × 1014 ions/cm2, and decreased at a fluence of 1 × 1015 ions/cm2. In the case of Ar+ ion beam-modified surfaces, the number of adhered endothelial cells increased as the fluence increased. Focusing on fluence, the number of adhered endothelial cells at a fluence of 1 × 1014 ions/cm2 showed no significant difference between the ion species. For a fluence of 5 × 1014 ions/cm2, the number of endothelial cells adhered onto Ar+ ion beam-modified surface showed the minimum value between other ion species. The number of adhered endothelial cells at a fluence of 1 × 1015 ions/cm2 slightly decreased as the mass number of the ion species increased.
2.3. Endothelial cell culture
2.5. Observation and analysis of surface morphology The surface morphology of the specimens was observed by means of SEM (JSM6330F, JEOL, Japan) after sputter-coating with gold in a plasma coater (SC-701, Sanyu Denshi Co., Ltd., Japan). The average crack size in the surfaces of the specimens was estimated with ImageJ software [12]. First, we converted the SEM image of the surface morphology of a specimen to an image segmented into black and white using a threshold function, and a median filter was applied to minimize the noise by calculating the average grayscale in a pixel from the values in the neighboring pixels. Then, the particle size corresponding to the crack in the surface was estimated with the ‘Analyze particles’ features of this software.
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
907
Control 10µm
He+
Ne+
Ar+
Kr+
1x1014 ions/cm2
5x1014 ions/cm2
1x1015 ions/cm2
Fig. 1. SEM images of human platelets adhered onto the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV, after contact with Ca2+-replenished PRP for 10 min.
3.3. Change in surface morphology The surfaces of non-modified and ion beam-modified ePTFE were observed by using SEM to examine the morphological changes in the ePTFE surface associated with ion beam modification. The nonmodified ePTFE had a micro-porous structure consisting of the submicron-sized nodes and fibrils of PTFE. After ion beam modification of the ePTFE surface, the node size decreased and the fibrils disappeared near the surface. Consequently, the pore size increased near the surface [9,11]. As a result, many cracks among the nodes were formed in ion beam-modified ePTFE surfaces, as seen in Fig. 1. In order to measure the average crack size in the ePTFE surfaces, the SEM images of the ePTFE surfaces were analyzed by using ImageJ software. The ImageJ software allows us to count the number of black pixels in each crack for every horizontal line of an image. However, many small pixels are in the SEM images of the ePTFE surfaces, which
have very fine structure, and therefore the average crack size in the ePTFE surfaces is estimated to be small. Fig. 5 shows the average crack size in a non-modified ePTFE surface as a control and in the ion beammodified ePTFE surfaces. After ion beam modification of the ePTFE surface, the average crack size increased in all of the surfaces of ion beam-modified ePTFE, compared to that in the non-modified ePTFE surface. The average crack size in He+, Ne+ and Ar+ ion beam-modified surfaces increased as the fluence increased. In the case of Kr+ ion beam-modified surfaces, the average crack size increased up to a fluence of 5 × 1014 ions/cm2, and decreased at a fluence of 1 × 1015 ions/cm2. Focusing on fluence, the average crack size at a fluence of 1 × 1014 ions/cm2 slightly increased as the mass number of the ion species increased. For the fluences of 5 × 1014 and 1 × 1015 ions/cm2, the average crack size in Ne+ ion beam-modified surfaces showed the maximum value between other ion species.
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
Number of adhered platelets [platelets/mm2]
908
4. Discussion
35 1×1014 ions/cm2
30
5×1014 ions/cm2
4.1. Platelet adhesion
1×1015 ions/cm2
25 20 15 10 5 0
Control
He+
Ne+
Ar+
Kr+
Fig. 2. Total number of platelets adhered onto the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV. These data are presented as mean ± standard deviation.
The foreign surfaces of artificial biomaterials for medical devices cause thrombus formation, which remains as one of the major problems in the development of blood-contacting materials. The thrombus formation on the surface is closely related to the surface properties such as surface energy, surface potential, surface morphology, surface chemical structure such as functional groups and characteristic groups, wettability (i.e. hydrophilicity and hydrophobicity) on the surface, and so on. Ion beam surface modification of ePTFE caused morphological changes in the surface, compared to the non-modified ePTFE surface. Comparing between Figs. 5 and 2, one can observe correlations between the average crack size and the total number of adhered platelets for ion beam-modified ePTFE surfaces. That is, the enlargement of the average crack size due to increasing the fluence for each ion species resulted in a decrease in the number of adhered platelets, and conversely, the reduction of the average crack size due to
Fig. 3. Morphology of BAECs cultured for 3 days on TCPS and the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV.
Number of adhered cells [x104 cells/ml]
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
20 18
1×1014 ions/cm2
Initial cell number
5×1014 ions/cm2
16
1×1015 ions/cm2
14 12 10 8 6 4 2 0
TCPS Control
He+
Ne+
Ar+
Kr+
Fig. 4. BAEC growth on TCPS and the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV. BAECs were inoculated with 3 × 104 cells/dish and the growth was monitored by counting cells over the course of 3 days. These data are presented as mean ± standard deviation.
increasing the fluence from 5 × 1014 to 1 × 1015 ions/cm2 for Kr+ ion resulted in an increase in the number of adhered platelets. However, for a fluence of 1 × 1014 ions/cm2, the slight enlargement of the average crack size due to increasing the mass number of the ion species resulted in a remarkable increase in the number of adhered platelets. This result is thought to be due to the influence of the change in the chemical structure of the surface layer of ePTFE associated with ion beam modification [9,11]. For the lower fluences such as 1 × 1014 ions/cm2, the radiation effects due to ion bombardment become greater for the heavier ions, and therefore the change in the chemical structure of the surface layer of ePTFE becomes greater for Kr+ ion beam modification. Therefore, it is thought that one of the reasons for the inhibition of platelet adhesion on ion beam-modified ePTFE surfaces is the enlargement of the crack size and the reduction of the node size in these surfaces, because of which the scaffolds for platelet adhesion would diminish in these surfaces. The inhibition of platelet adhesion, activation and aggregation has the potential to improve anti-thrombogenicity on the surface.
the surface, compared to that on the non-modified ePTFE surface, as shown in Fig. 4. In comparison between Figs. 5 and 4, there was no correlation between the average crack size and the total number of adhered endothelial cells for ion beam-modified ePTFE surfaces. However, compared to the non-modified ePTFE surface, ion beam surface modification of ePTFE caused the significant morphological changes in the surface, and endothelial cell adhesion, spreading and proliferation were significantly promoted on the ion beam-modified ePTFE surfaces. Ion beam surface modification of ePTFE also caused a change in the chemical structure of the surface layer; specifically, \CF2\ bonds of ePTFE were degraded, and NC_O, NC_Cb and \CF3 bonds were newly produced in the surface layer [9,11]. Therefore, it is thought that the surface roughness, carbonyl groups and unsaturated bonds in the ePTFE surface associated with ion beam modification are effective in cell adhesion, and therefore ion beam-modified ePTFE surfaces would enhance the endothelium lining due to the improvement of endothelial cell adhesion, spreading and proliferation. 4.3. Surface morphology Ion beam surface modification of ePTFE caused an increase in the average crack size in the surface, compared to that in the non-modified ePTFE surface, as shown in Fig. 5. However, conversely, the average crack size at the fluences of 5 × 1014 and 1 × 1015 ions/cm2 decreased as the mass number of the bombarded-ion increased from Ne+ to Kr+ ion. Also, the average crack size in Kr+ ion beam-modified surfaces decreased as the fluence increased from 5 × 1014 to 1 × 1015 ions/cm2. The cross-sectional images of ion beam-modified ePTFE surfaces indicate that ion beam surface modification of ePTFE formed spinelike nodes perpendicular to the surface rather than cracks [9,11]. Therefore, it is thought that the ridges of the spine-like nodes in the surfaces of ion beam-modified ePTFE are etched by ion bombardment by which the radiation effects become greater for the heavier ions or the higher fluences, and therefore the node size increases and the crack size decreases in these surfaces. 5. Conclusion
4
The surface of ePTFE was modified with ion beam irradiation to improve the blood compatibility, which was evaluated by in vitro platelet adhesion and endothelial cell adhesion tests. The surface modification of ePTFE sheets utilizing He+, Ne+, Ar+ and Kr+ ion beams was performed at an energy of 150 keV with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2. Platelet response was inhibited on the surfaces modified with He+, Ne+ and Ar+: 5 × 1014 and 1 × 1015 ions/cm2, and Kr+: 5 × 1014 ions/cm2; however, platelet response was promoted on the surfaces modified with He+, Ne+ and Ar+: 1 × 1014 ions/cm2, and Kr+: 1 × 1014 and 1 × 1015 ions/cm2, compared to the non-modified ePTFE surface. Although the nonmodified ePTFE surface dramatically inhibited endothelial cell growth, this growth was significantly promoted on all of the surfaces of ion beam-modified ePTFE. These results suggest that the surfaces of ion beam-modified ePTFE have the potential to enhance the formation of neointima (i.e. pseudointima) by endothelium lining through not only the promotion of endothelial cell growth but also the inhibition of platelet response. The neointima formed by endothelium lining brings about anti-thrombogenicity on the surface.
2
References
4.2. Endothelial cell adhesion Ion beam surface modification of ePTFE caused the powerful promotion of endothelial cell adhesion, spreading and proliferation on
16 1×1014 ions/cm2
14 Average crack size [µm 2]
909
5×1014 ions/cm2 1×1015 ions/cm2
12 10 8 6
0
Control
He+
Ne+
Ar+
Kr+
Fig. 5. Average crack size in the ePTFE surfaces: non-modification as a control, and ion beam modification using He+, Ne+, Ar+ and Kr+ ions with fluences of 1 × 1014, 5 × 1014 and 1 × 1015 ions/cm2 at an energy of 150 keV.
[1] H. Matsumoto, T. Hasegawa, K. Fuse, M. Yamamoto, M. Saigusa, Surgery 74 (1973) 519. [2] S. Yamagata, K. Goto, Y. Oda, H. Kikuchi, Neurol. Med. Chir. Tokyo 33 (1993) 582. [3] G. Bhatnagar, S.E. Fremes, G.T. Christakis, B.S. Goldman, J. Card. Surg. 13 (1998) 190. [4] R.A. Monaghan, S. Meban, Can. J. Surg. 34 (1991) 502.
910
H. Hiruma et al. / Surface & Coatings Technology 206 (2011) 905–910
[5] Y. Kawahira, T. Yagihara, H. Uemura, T. Ishizaka, K. Yoshizumi, S. Kitamura, Eur. J. Cardiothorac. Surg. 15 (1999) 289. [6] J. Gottlow, S. Nyman, J. Lindhe, T. Karring, J. Wennström, J. Clin. Periodontol. 13 (1986) 604. [7] G. Setzen, E.F. Williams, Plast. Reconstr. Surg. 100 (1997) 1788. [8] N. Takahashi, H. Ujiie, Y. Suzuki, M. Iwaki, T. Hori, No Shinkei Geka 32 (2004) 339. [9] Y. Suzuki, M. Iwaki, S. Tani, G. Oohashi, M. Kamio, Nucl. Instrum. Methods Phys. Res. B 206 (2003) 538.
[10] N. Takahashi, Y. Suzuki, H. Ujiie, T. Hori, M. Iwaki, T. Yamada, Nucl. Instrum. Methods Phys. Res. B 242 (2006) 61. [11] N. Takahashi, Y. Suzuki, H. Ujiie, M. Iwaki, T. Hori, T. Yamada, Surf. Coat. Technol. 201 (2007) 8150. [12] U.S. National Institutes of Health, World Wide Web electronic publication, http:// rsbweb.nih.gov/ij/.