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Negative-ion beam surface modification of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties
Nuclear Instruments and Methods in Physics Research B 148 (1999) 1136±1140
Negative-ion beam surface modi®cation of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties H. Tsuji *, H. Satoh, S. Ikeda, S. Ikemura, Y. Gotoh, J. Ishikawa Department of Electronic Science and Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan
Abstract Negative-silver-ion implantation into tissue-culture polystyrene (TCPS) dishes was investigated and it was found to modify hydrophilic and cell attachment properties of the dishes. Negative-ion implantation has an advantage of being almost free of surface charging, and is a suitable method for implantation into insulators such as polymers. Negative silver ions are used due to the antibacterial property of silver. Ag-implanted TCPS dishes had a contact angle larger than the normal value of 66° of unimplanted dishes. The contact angle of water had a strong dependence on the ion energy rather than the dose. As a cell-culture experiment, human umbilical vascular endothelial cell (HUVEC) was used in unimplanted and Ag-implanted TCPS dishes, the implantation removed the cell-attachment property of the surface. In implantation with a mask with a striped pattern, most attached cells of HUVEC were in the unimplanted region aligned along a stripe direction. Ó 1999 Elsevier Science B.V. All rights reserved. PACS: 81.60.-h; 68.10.Cr; 68.90.+g; 87.73.-w Keywords: Surface modi®cation; Negative-ion implantation; Biocompatibility; Polymers
1. Introduction The ion implantation technique is a promising method for surface modi®cation of biomedical polymers to improve and control biocompatibility for blood and cell attachment [1±4], as well as modi®cation of tribological, electrical and optical properties [5±7]. Progress of research on surface modi®cation of polymers by ion implantation is
* Corresponding author. Tel.: +81 75 7535354; fax: +81 75 7511576; e-mail: [email protected]
expected in the biomedical ®eld for improving biocompatibility and for conveying new biofunctionabilitites such as antibacterial, selective adhesion and cell-orientation properties. Negative-ion implantation brings us almost ``charge-up free'' implantation into isolated electrodes and insulators [8,9] without external charge compensation, where conventional positive-ion implantation causes a charge-up problem which aects the dose and the implantation energy of ions. In our previous researches [3,4], we implanted silver negative ions into polystyrene dishes (PS) at a low energy of about 10 keV, and showed
0168-583X/98/$ ± see front matter Ó 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 8 0 2 - 7
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
1137
that the surface of PS was modi®ed to have a low contact angle and to have good cell attachment properties. Tissue-culture polystyrene dishes (TCPS) are usually used in cell-culture. The surface is made to have hydrophilic properties for tissue culture. In this study, the modi®cation the properties of TCPS by negative-silver-ion implantation was investigated with respect to surface wettability and cell-attachment properties of human umbilical vascular endothelial cells (HUVEC). In addition, we investigated eects of using micro-size patterned implantation through a mask.
water of a diameter of 0.8±1.2 mm (0.3±0.9 ll) was put on an area of the Ag-implanted surface. Pictures of the droplets through an optical microscope were taken from a horizontal direction promptly after the placement. To prevent the evaporation eect of water, which makes the contact angle small, we put other large droplets of water around the examined area in the sample dish and a cover on the dish. The shape of droplets was con®rmed to be constant within experimental error in constant humidity and at a temperature of 20°C. Contact angles were calculated from width and height of the droplet, and more than six measurements were averaged for each sample.
2. Experimental
2.3. Cell culture of HUVEC
2.1. Negative-silver-ion implantation
Human umbilical vascular endothelial cells (HUVEC) were collected from an umbilical vein by the method of Jae et al. [12]. Cells were used as a seed after incubation in gelatin-coated tissueculture polystyrene ¯asks with Medium 199 containing supplements of 15% fetal bovine serum, heparin, endothelial cell growth supplements, Lglutamine and antibiotics. Polystyrene dishes after Ag-implantation were sterilized for one day in 70% ethanol, which was replaced by sterilized deionized water three times over another two days. A con¯uent amount (4± 5 ´ 104 cells per cm2 ) of HUVEC cells were plated on Ag-implanted TCPS dishes and cultured at 37°C in 5% CO2 in air for 3 weeks with Medium 199 containing the supplements. The culture media were changed every three days. HUVEC growth and attachment to the dish surfaces were evaluated by counting the attached cell number for an area of 1 mm2 under a phase contrast microscope after periods of culture. At the end of the culture we observed attached cells with an optical microscope after Giemsa staining [13].
Tissue-culture polystyrene dishes (TCPS, Corning, 6 cm in diameter, No. 25010-60) were implanted with negative-silver ions in a negativeion implanter system [10]. The ions (Agÿ ) were produced from a silver (99.9%) sputtering target by sputtering with cesium ions in a neutral and ionized alkaline bombardment-type heavy negative-ion source, NIABNIS [11]. After mass-separation, the beam of Agÿ ions entered the sample surface through a limiting hole of 11.28 mm diameter in a Faraday cup. Thus, TCPS dishes were implanted with Agÿ ions in an area of 1 cm2 . The implantation energy was 5, 10, 20, or 30 keV and the dose was in a range from 1.0 ´ 1014 to 3.0 ´ 1016 ions/cm2 with a current density of 30 to 300 nA/ cm2 , depending on the ion energy. Residual and background gas pressures were about 1.0 ´ 10ÿ3 and 1.4 ´ 10ÿ4 Pa, respectively. For patterned implantation, we used a nickel thin plate as a mask with a striped pattern which had slits of 40 lm-wide and 4 mm-long with a spacing of 40 lm. Negative-silver ions were implanted through this mask at an energy of 20 keV with a dose of 3 ´ 1015 ions/cm2 .
3. Results and discussion
2.2. Contact angle
3.1. Change of hydrophilic properties
For measuring contact angles for evaluation of the hydrophilic properties, a spherical drop of pure
The contact angle of water to the surface of tissue-culture polystyrene (TCPS) dish increases
1138
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
after ion implantation. Fig. 1 shows contact angles of Ag-implanted TCPS at a dose of 3.0 ´ 1015 ions/ cm2 as a function of the ion energy. The contact angle increased signi®cantly from 72° to 82° with an increase in ion energy in the range from 5 to 30 keV, while the unimplanted TCPS has a contact angle of 66°. As for the dependence on ion dose, the contact angle increased with an increase in ion dose. This increase of contact angle is considered to be due to reduction by the bombardment during ion implantation of functional groups related to the hydrophilic properties. Thus, the ion implantation modi®es the surface hydrophilic properties of TCPS. 3.2. Cell-attachment and growth properties Fig. 2 shows micrographs of human umbilical vascular endothelial cells (HUVEC) on Ag-implanted TCPS dishes at ion energies of (a) 10 keV and (b) 30 keV with Giemsa staining after culturing for 21 days. In the micrographs, many HUVEC cells have attached to the unimplanted surface with spreading pseudopodia. A few were observed on the Ag-implanted area of the 10 keVimplanted TCPS, but none for 30 keV-implanted
Fig. 2. Micrographs of human umbilical vascular endothelial cell (HUVEC) on TCPS dishes (dose of 3 ´ 1015 ions/cm2 ) at ion energies of (a) 10 keV and (b) 30 keV after 21 days in cell culture.
Fig. 1. Contact angle of Ag-implanted TCPS as a function of the ion energy at an ion dose of 3 ´ 1015 ions/cm2 .
TCPS. In general, the unimplanted TCPS had good cell-attachment properties. Fig. 3 shows the number of HUVEC cells attached to a 1 mm2 area as a function of the culture period for various implanted and unimplanted TCPS surfaces. The number of HUVEC cells attached to the unimplanted TCPS remained at more than 400 cells after 21 days, and this number was comparable to the seeded number. On the Ag-implanted polystyrene, the number of HUVEC decreased rapidly to zero after 3 days, except for the 5 keV-implanted TCPS. In Fig. 4, the number of HUVEC cells attached to the surface after culture periods of 2 and 5 days
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
Fig. 3. Number of HUVEC cells attached to various AG-implanted TCPS surfaces in as a function of the culture day until 21 days. The parameters are implantation energy of negativesilver ions with a dose of 3 ´ 1015 ions/cm2 : 5, 10, 20, and 30 keV. As a control, the data for unimplanted TCPS are also shown.
1139
polystyrene obtained in the previous work [4] is also plotted in Fig. 4 for the reference. The threshold contact angle for cell-adhesion was found to be about 76°. Surfaces with contact angle less than this value showed cell-attachment properties. Fig. 5 shows attached HUVEC cells on the stripe patterned TCPS by Ag-implantation through the mask (40 lm-wide and 4 mm-long with 40 lm-spacing). Cells attached to the unimplanted region, as expected from the result mentioned above. In particular, the cells seemed to be aligned along the long direction of the stripe pattern. The reason of this cell-orientation property was not understood yet. But, this demonstrates that the ion implantation with a micro-sized (a little less than the size of cell, i.e. several tens of lm) pattern could bring a new biofunctionability. 4. Conclusions
is plotted versus the contact angle, taking data from Figs. 1 and 3. The data for unimplanted
Negative-silver ions were implanted in TCPS dishes to change biocompatibility. The contact angle of TCPS surfaces was increased by the ion implantation with increased ion energy in a range below 30 keV, and 82° was obtained for the sur-
Fig. 4. Change of the number of HUVEC cells attached to various TCPS surfaces after for 2 and 5 days in relation to the contact angle. In the ®gure, the data for the unimplanted normal polystyrene (Ref. [4]) are also shown as a reference.
Fig. 5. Micrograph of HUVEC attached to the surface of the TCPS Ag-implanted at 20 keV with a dose of 3 ´ 1015 ions/cm2 through a mask with stripe pattern 40 lm wide and 4 mm long at 40 lm spacing.
1140
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
face Ag-implanted at 30 keV with 3 ´ 1015 ions/ cm2 . By cell culture of HUVEC, cell-attachment properties were observed to be changed by the implantation. Negative-silver-ion implanted TCPS surfaces had drastically reduced HUVEC attachment. The eect on the antibacterial property of silver atoms was not examined in these experiments. A critical relation between cell-attachment properties and contact angle was observed. A critical contact angle of 76° was identi®ed. In the case of patterning implantation with the striped mask, a new property of cell orientation was found. Cells were aligned along a long direction of the unimplanted region with 40 lm in width.
References [1] Y. Suzuki, M. Kusakabe, K. Kusakabe, H. Akiba, M. Iwaki, Nucl. Instr. and Meth. B 59/60 (1991) 698. [2] Y. Suzuki, M. Kusakabe, J.S. Lee, M. Kusakabe, M. Iwaki, H. Sasabe, Nucl. Instr. and Meth. B 65 (1992) 142.
[3] H. Tsuji, H. Satoh, S. Ikeda, Y. Gotoh, J. Ishikawa, Nucl. Instr. and Meth. B 141 (1998) 197. [4] H. Tsuji, H. Satoh, S. Ikeda, N. Ikemoto, Y. Gotoh, J. Ishikawa, Surface and Coatings Technology 103/104 (1998) 124. [5] G. Marletta, Nucl. Instr. and Meth. B 46 (1990) 295. [6] G. Foti, R. Reitano, Nucl. Instr. and Meth. B 46 (1990) 306. [7] J. Davenas, P. Thevenard, G. Boiteux, M. Fallavier, X.L. Lu, Nucl. Instr. and Meth. B 46 (1990) 317. [8] H. Tsuji, Y. Toyota, J. Ishikawa, S. Sakai, Y. Okayama, S. Nagumo, Y. Gotoh, K. Matsuda, Ion Implantation Technology-94, Elsevier, New York, 1995, p. 612. [9] S. Sakai, Y. Gotoh, H. Tsuji, Y. Toyata, J. Ishikawa, M. Tanjyo, K. Matsuda, Nucl. Instr. and Meth. B 96 (1995) 43. [10] J. Ishikawa, H. Tsuji, Nucl. Instr. and Meth. B 74 (1993) 118. [11] H. Tsuji, T. Taya, J. Ishikawa, T. Takagi, in: T. Takagi (Ed.), Proceedings of the International Ion Engineering Congress, ISIAT'83 and IPAT'83, Institute of Electrical Engineers of Japan, vol. 1, 12±19 September 1983, Kyoto, pp. 141±146. [12] E.A. Jae, R.L. Nachman, C.G. Becker, C.R. Minick, J. Clin. Invest. 52 (1973) 2745. [13] K. Imahori, T. Yamakawa (Eds.), Dictionary of Biochemistry (in Japanese), Tokyo Kagaku Doujin, 1988, p. 311.
Negative-ion beam surface modi®cation of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties H. Tsuji *, H. Satoh, S. Ikeda, S. Ikemura, Y. Gotoh, J. Ishikawa Department of Electronic Science and Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan
Abstract Negative-silver-ion implantation into tissue-culture polystyrene (TCPS) dishes was investigated and it was found to modify hydrophilic and cell attachment properties of the dishes. Negative-ion implantation has an advantage of being almost free of surface charging, and is a suitable method for implantation into insulators such as polymers. Negative silver ions are used due to the antibacterial property of silver. Ag-implanted TCPS dishes had a contact angle larger than the normal value of 66° of unimplanted dishes. The contact angle of water had a strong dependence on the ion energy rather than the dose. As a cell-culture experiment, human umbilical vascular endothelial cell (HUVEC) was used in unimplanted and Ag-implanted TCPS dishes, the implantation removed the cell-attachment property of the surface. In implantation with a mask with a striped pattern, most attached cells of HUVEC were in the unimplanted region aligned along a stripe direction. Ó 1999 Elsevier Science B.V. All rights reserved. PACS: 81.60.-h; 68.10.Cr; 68.90.+g; 87.73.-w Keywords: Surface modi®cation; Negative-ion implantation; Biocompatibility; Polymers
1. Introduction The ion implantation technique is a promising method for surface modi®cation of biomedical polymers to improve and control biocompatibility for blood and cell attachment [1±4], as well as modi®cation of tribological, electrical and optical properties [5±7]. Progress of research on surface modi®cation of polymers by ion implantation is
* Corresponding author. Tel.: +81 75 7535354; fax: +81 75 7511576; e-mail: [email protected]
expected in the biomedical ®eld for improving biocompatibility and for conveying new biofunctionabilitites such as antibacterial, selective adhesion and cell-orientation properties. Negative-ion implantation brings us almost ``charge-up free'' implantation into isolated electrodes and insulators [8,9] without external charge compensation, where conventional positive-ion implantation causes a charge-up problem which aects the dose and the implantation energy of ions. In our previous researches [3,4], we implanted silver negative ions into polystyrene dishes (PS) at a low energy of about 10 keV, and showed
0168-583X/98/$ ± see front matter Ó 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 8 0 2 - 7
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
1137
that the surface of PS was modi®ed to have a low contact angle and to have good cell attachment properties. Tissue-culture polystyrene dishes (TCPS) are usually used in cell-culture. The surface is made to have hydrophilic properties for tissue culture. In this study, the modi®cation the properties of TCPS by negative-silver-ion implantation was investigated with respect to surface wettability and cell-attachment properties of human umbilical vascular endothelial cells (HUVEC). In addition, we investigated eects of using micro-size patterned implantation through a mask.
water of a diameter of 0.8±1.2 mm (0.3±0.9 ll) was put on an area of the Ag-implanted surface. Pictures of the droplets through an optical microscope were taken from a horizontal direction promptly after the placement. To prevent the evaporation eect of water, which makes the contact angle small, we put other large droplets of water around the examined area in the sample dish and a cover on the dish. The shape of droplets was con®rmed to be constant within experimental error in constant humidity and at a temperature of 20°C. Contact angles were calculated from width and height of the droplet, and more than six measurements were averaged for each sample.
2. Experimental
2.3. Cell culture of HUVEC
2.1. Negative-silver-ion implantation
Human umbilical vascular endothelial cells (HUVEC) were collected from an umbilical vein by the method of Jae et al. [12]. Cells were used as a seed after incubation in gelatin-coated tissueculture polystyrene ¯asks with Medium 199 containing supplements of 15% fetal bovine serum, heparin, endothelial cell growth supplements, Lglutamine and antibiotics. Polystyrene dishes after Ag-implantation were sterilized for one day in 70% ethanol, which was replaced by sterilized deionized water three times over another two days. A con¯uent amount (4± 5 ´ 104 cells per cm2 ) of HUVEC cells were plated on Ag-implanted TCPS dishes and cultured at 37°C in 5% CO2 in air for 3 weeks with Medium 199 containing the supplements. The culture media were changed every three days. HUVEC growth and attachment to the dish surfaces were evaluated by counting the attached cell number for an area of 1 mm2 under a phase contrast microscope after periods of culture. At the end of the culture we observed attached cells with an optical microscope after Giemsa staining [13].
Tissue-culture polystyrene dishes (TCPS, Corning, 6 cm in diameter, No. 25010-60) were implanted with negative-silver ions in a negativeion implanter system [10]. The ions (Agÿ ) were produced from a silver (99.9%) sputtering target by sputtering with cesium ions in a neutral and ionized alkaline bombardment-type heavy negative-ion source, NIABNIS [11]. After mass-separation, the beam of Agÿ ions entered the sample surface through a limiting hole of 11.28 mm diameter in a Faraday cup. Thus, TCPS dishes were implanted with Agÿ ions in an area of 1 cm2 . The implantation energy was 5, 10, 20, or 30 keV and the dose was in a range from 1.0 ´ 1014 to 3.0 ´ 1016 ions/cm2 with a current density of 30 to 300 nA/ cm2 , depending on the ion energy. Residual and background gas pressures were about 1.0 ´ 10ÿ3 and 1.4 ´ 10ÿ4 Pa, respectively. For patterned implantation, we used a nickel thin plate as a mask with a striped pattern which had slits of 40 lm-wide and 4 mm-long with a spacing of 40 lm. Negative-silver ions were implanted through this mask at an energy of 20 keV with a dose of 3 ´ 1015 ions/cm2 .
3. Results and discussion
2.2. Contact angle
3.1. Change of hydrophilic properties
For measuring contact angles for evaluation of the hydrophilic properties, a spherical drop of pure
The contact angle of water to the surface of tissue-culture polystyrene (TCPS) dish increases
1138
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
after ion implantation. Fig. 1 shows contact angles of Ag-implanted TCPS at a dose of 3.0 ´ 1015 ions/ cm2 as a function of the ion energy. The contact angle increased signi®cantly from 72° to 82° with an increase in ion energy in the range from 5 to 30 keV, while the unimplanted TCPS has a contact angle of 66°. As for the dependence on ion dose, the contact angle increased with an increase in ion dose. This increase of contact angle is considered to be due to reduction by the bombardment during ion implantation of functional groups related to the hydrophilic properties. Thus, the ion implantation modi®es the surface hydrophilic properties of TCPS. 3.2. Cell-attachment and growth properties Fig. 2 shows micrographs of human umbilical vascular endothelial cells (HUVEC) on Ag-implanted TCPS dishes at ion energies of (a) 10 keV and (b) 30 keV with Giemsa staining after culturing for 21 days. In the micrographs, many HUVEC cells have attached to the unimplanted surface with spreading pseudopodia. A few were observed on the Ag-implanted area of the 10 keVimplanted TCPS, but none for 30 keV-implanted
Fig. 2. Micrographs of human umbilical vascular endothelial cell (HUVEC) on TCPS dishes (dose of 3 ´ 1015 ions/cm2 ) at ion energies of (a) 10 keV and (b) 30 keV after 21 days in cell culture.
Fig. 1. Contact angle of Ag-implanted TCPS as a function of the ion energy at an ion dose of 3 ´ 1015 ions/cm2 .
TCPS. In general, the unimplanted TCPS had good cell-attachment properties. Fig. 3 shows the number of HUVEC cells attached to a 1 mm2 area as a function of the culture period for various implanted and unimplanted TCPS surfaces. The number of HUVEC cells attached to the unimplanted TCPS remained at more than 400 cells after 21 days, and this number was comparable to the seeded number. On the Ag-implanted polystyrene, the number of HUVEC decreased rapidly to zero after 3 days, except for the 5 keV-implanted TCPS. In Fig. 4, the number of HUVEC cells attached to the surface after culture periods of 2 and 5 days
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
Fig. 3. Number of HUVEC cells attached to various AG-implanted TCPS surfaces in as a function of the culture day until 21 days. The parameters are implantation energy of negativesilver ions with a dose of 3 ´ 1015 ions/cm2 : 5, 10, 20, and 30 keV. As a control, the data for unimplanted TCPS are also shown.
1139
polystyrene obtained in the previous work [4] is also plotted in Fig. 4 for the reference. The threshold contact angle for cell-adhesion was found to be about 76°. Surfaces with contact angle less than this value showed cell-attachment properties. Fig. 5 shows attached HUVEC cells on the stripe patterned TCPS by Ag-implantation through the mask (40 lm-wide and 4 mm-long with 40 lm-spacing). Cells attached to the unimplanted region, as expected from the result mentioned above. In particular, the cells seemed to be aligned along the long direction of the stripe pattern. The reason of this cell-orientation property was not understood yet. But, this demonstrates that the ion implantation with a micro-sized (a little less than the size of cell, i.e. several tens of lm) pattern could bring a new biofunctionability. 4. Conclusions
is plotted versus the contact angle, taking data from Figs. 1 and 3. The data for unimplanted
Negative-silver ions were implanted in TCPS dishes to change biocompatibility. The contact angle of TCPS surfaces was increased by the ion implantation with increased ion energy in a range below 30 keV, and 82° was obtained for the sur-
Fig. 4. Change of the number of HUVEC cells attached to various TCPS surfaces after for 2 and 5 days in relation to the contact angle. In the ®gure, the data for the unimplanted normal polystyrene (Ref. [4]) are also shown as a reference.
Fig. 5. Micrograph of HUVEC attached to the surface of the TCPS Ag-implanted at 20 keV with a dose of 3 ´ 1015 ions/cm2 through a mask with stripe pattern 40 lm wide and 4 mm long at 40 lm spacing.
1140
H. Tsuji et al. / Nucl. Instr. and Meth. in Phys. Res. B 148 (1999) 1136±1140
face Ag-implanted at 30 keV with 3 ´ 1015 ions/ cm2 . By cell culture of HUVEC, cell-attachment properties were observed to be changed by the implantation. Negative-silver-ion implanted TCPS surfaces had drastically reduced HUVEC attachment. The eect on the antibacterial property of silver atoms was not examined in these experiments. A critical relation between cell-attachment properties and contact angle was observed. A critical contact angle of 76° was identi®ed. In the case of patterning implantation with the striped mask, a new property of cell orientation was found. Cells were aligned along a long direction of the unimplanted region with 40 lm in width.
References [1] Y. Suzuki, M. Kusakabe, K. Kusakabe, H. Akiba, M. Iwaki, Nucl. Instr. and Meth. B 59/60 (1991) 698. [2] Y. Suzuki, M. Kusakabe, J.S. Lee, M. Kusakabe, M. Iwaki, H. Sasabe, Nucl. Instr. and Meth. B 65 (1992) 142.
[3] H. Tsuji, H. Satoh, S. Ikeda, Y. Gotoh, J. Ishikawa, Nucl. Instr. and Meth. B 141 (1998) 197. [4] H. Tsuji, H. Satoh, S. Ikeda, N. Ikemoto, Y. Gotoh, J. Ishikawa, Surface and Coatings Technology 103/104 (1998) 124. [5] G. Marletta, Nucl. Instr. and Meth. B 46 (1990) 295. [6] G. Foti, R. Reitano, Nucl. Instr. and Meth. B 46 (1990) 306. [7] J. Davenas, P. Thevenard, G. Boiteux, M. Fallavier, X.L. Lu, Nucl. Instr. and Meth. B 46 (1990) 317. [8] H. Tsuji, Y. Toyota, J. Ishikawa, S. Sakai, Y. Okayama, S. Nagumo, Y. Gotoh, K. Matsuda, Ion Implantation Technology-94, Elsevier, New York, 1995, p. 612. [9] S. Sakai, Y. Gotoh, H. Tsuji, Y. Toyata, J. Ishikawa, M. Tanjyo, K. Matsuda, Nucl. Instr. and Meth. B 96 (1995) 43. [10] J. Ishikawa, H. Tsuji, Nucl. Instr. and Meth. B 74 (1993) 118. [11] H. Tsuji, T. Taya, J. Ishikawa, T. Takagi, in: T. Takagi (Ed.), Proceedings of the International Ion Engineering Congress, ISIAT'83 and IPAT'83, Institute of Electrical Engineers of Japan, vol. 1, 12±19 September 1983, Kyoto, pp. 141±146. [12] E.A. Jae, R.L. Nachman, C.G. Becker, C.R. Minick, J. Clin. Invest. 52 (1973) 2745. [13] K. Imahori, T. Yamakawa (Eds.), Dictionary of Biochemistry (in Japanese), Tokyo Kagaku Doujin, 1988, p. 311.