958

COMMUNICATION

Selective adhesion and proliferation of cells on ion-implanted polymer domains

Jae-Suk Lee, Makoto Kaibara, Masaya Iwaki, Hiroyuki Sasabe, Yoshiaki Suzuki* and bask Kusakabe* The lnsfifut~ of Physical and &hem~ca~ Research ~~IKEff~, 2-7 ffirosawa, Wake, Saifama 357-01, Japan; “Sony Corporation, 6-7-35 Kifashinagawa, Shinagawa-ku, Tokyo 741, Japan

We have found that the adhesion and proliferation of endothelial cells can be drastically improved when cultivated on an ion-implanted polymer surface. When the surface of segmented polyurethane, where endothelial cells are not capable of proliferating, is modified by Ne+ or Na” ion implantation with a fluence of 1 x lOi ions/cm’ at an energy of 150 keV, cell adhesion and proliferation occurred selectively on the ion-implanted region irrespective of the ion species. The cells did not proliferate at ion fluences below 1 x lOi ions/cm2. Most cells migrated into the ion-implanted domain within l-2 h, but some of the cells attached outside of the region and then slowly migrated into the region. Ion implantation of polystyrene, on which cells are capable of proliferating, further promoted cell spreading and proliferation, and increased resistance to detachment when the cells were exposed to trypsin.

Keywords: Cell adhesioff, endofhefjal celts, ~ol~urefhanes

Received 28 August 1992; revised 7 October 1992; accepted 5 May 1993

Ion implantation is an important technique for implant- ing impurities in the manufacturing process of semiconductors. Ion implantation has also been shown to be a useful technique for improving the surface properties of metals, such as wear and corrosion resistance’. In recent years, ion implantation has been used in the modification of polymers’, including biocompatible polymers3.

Attachment, spreading and proliferation of anchorage- dependent biological cells are influenced by surface structures or properties of the substrates. Endothelial cells are not capable of proliferating on polymers such as polyurethane. We present here the selective adhesion and growth of endothelial cells on an ion- implanted segmented polyurethane surface.

MATERIALS AND METHODS

Polymers used were polystyrene (PSt) (culture dish, Falcon 1008, Becton-Dickinson, NJ, USA) and seg- mented polyurethane (SPU). SPU was obtained from Kanegafuchi Chemical Industry (Osaka, Japan). It contains about 13% poly(ethylene oxide)- poly(dimethylsiloxane)-poly(ethylene oxide) (PES),

Correspondence to Dr J.-S. Lee.

which is an active hydrogen compound. SPU was dissolved in tetrahydrofuran (Kant0 Chemicals, Tokyo, Japan) in the concen~ation of 5gflOOml. The solution was coated onto the inner surface of a glass dish (inner diameter of 2.8cm) and then the surface was dried under a gentle N2 gas stream.

Nef or Nat ion implantations were performed on a mask-covered surface at an energy of 150 keV, with a fluence of 1 x 1Ol5 ions/cm’, at a current lower than 0.5 PA/cm2 and at room temperature.

Bovine aorta endothelial cells (BAECs) were isolated from a descending thoracic aorta using 0.1% colla- genase b

K a method adapted from Jaffe et ~1.~ and

Schwartz . BAECs (5 x lo4 or 2 x lo* cells/ml) were suspended in medium (RPM1 1640, Nissui Pharmaceu- ticals, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). This cell suspension was poured onto the ion- implanted surface of the dish and incubated for 1-5 d at 37°C in a humidified atmosphere of 5% CO2 in air.

The cells were observed with an IMT-2 phase contrast microscope (Olympus, Tokyo, Japan). The cells were also examined using a system which consists of the phase contrast microscope, a TV camera (Model CTC-50OOJS, Ikegami Tsushinki, Tokyo, Japan), a TV monitor and a video recorder, in a humidified atmosphere of 5% CO2 in air at 37°C.

Biomaterials 1993, VoI. 14 No. 12 $2 1953 Butterworth-Heinemann Ltd 0142-~12/93/12~~-03

Selective cell adhesion and proliferation: J.-S. Lee et at. 959

RESULTS AND DISCUSSION

Figure 1 shows microscopic observations of BAECs which have proliferated on SPU within the Ne+ ion- implanted domains (inside circles). BAECs are not capable of proliferating on SPU (Figure la), but BAECs have selectively adhered (Figure ~b) and proliferated (Figure IC) on the ion-implanted domains. The average cell number in an ion-implanted domain increased from 2.8 x lo4 cells/cm2 at 1 d after seeding to 9.3 x lo4 cells/cm2 at 5 d. It is clear that cell adhesion and proliferation on SPU were drastically improved by ion implantation.

Figure z shows the percentage of cells which have entered into the ion-implanted domain versus the time after seeding. The number of cells in 10 ion-implanted domains (area of one domain = 3.1 x lo4 pm’) and the number outside of the ion-implanted domains were counted. The total area of cell counting, including ion- implanted domains, was 6.3 x 10” pm’. The ion- implanted area was 50% of the total area. About 95% of seeded cells entered into the domain within 2-3 h, but some cells attached outside of the domain and then moved slowly into the domain and spread after several hours.

Figure 3 shows the proliferation of BAECs on PSt within Nat ion-implanted domains (inside circles) at

Figure 1 BAEC growth on a, non-implanted SPU, and on Ne+ ion-implanted SPU domains (inside the circles; diameter of a circle is 200pm) at b, 1 d and c, 5d after seeding (bar = lOO/dm). The initial cell number seeded was about 5 x lo4 cells/ml.

100 - r I A I

T T 80 -

3

Q

: 60 c

40 - .I,

OT' I I I I 1 0 1 2 3 4 5 6 7

Time (h)

Figure 2 Percentage of cells entered into Na’ ion- implanted SPU domains (100 x n/n,; ntl is total cell number counted and n the entered cell number) plotted against the time. The initial cell number seeded was about 5 x 104cells/ml. Each mean is the average of six indepen- dent experiments.

Figure 3 BAECs on Na+ eon-implanted (inside circles) PSt dish at a, 2d and b, 5d after seeding (bar = 1OO~m~. The initial number of BAECs was 2 x 10 cells/ml. c, Trypsin- treated BAECs on ion-implanted (upper part) and non- implanted (lower part) PSt surface at Sd after seeding.

2 d (Figure3a) and 5 d (Figure3h) after seeding. It is clear that cell density at 5 d in the ion-implanted domains is higher than that in non-implanted domains. BAECs are capable of proliferating on non- implanted PSt, but cells seem to migrate willingly into the ion-implanted domains and then proliferate in those regions. At 2 d, cells in the ion-implanted domains adhered with a spread-out form, whereas cells in the non-implanted regions were round in shape.

Figure 3c shows cell detachment from the PSt surface when the BAEC monolayer was exposed to the trypsin solution. Trypsin (0.125%) (Difco, MI, USA) was dissolved in phosphate buffered saline (PBS) and put in contact with the BAEC monolayer for 2 min at 25°C. The trypsin solution was then removed, and the cells were rinsed once with PBS prior to the microscopic observation and cell counting. Most cells in the non- implanted region easily detached from the surface, but a number of cells still remained on the ion-implanted surface. Their shapes, however, changed to the round shape. Perhaps when the cells are exposed to trypsin, the cells are more resistant to detachment from the ion-implanted surface than from the non-implanted surface. It is assumed that ion implantation brought about the increase in resistance to the detachment of cells.

When ion implantation of SPU was performed with different ion species at an energy of 150keV with a fluence of 1 x 1015 ions/cm’, cells were still capable of proliferating on the ion-implanted regions, irrespec- tive of the ion species used (Nat., Ari, Ne’, IQ?, N:, and 0;). When Nat- ion implantation was performed on SPU at an energy of 150 keV with fluences between 1 x 1o13 and 1 x 1017ions/cm2, cell proliferation occurred with the fluence above 1 x 10*5ionslcm2. Also, cell proliferation on Nat ion-implanted SPU occurred at energies of 50, 100 and 150 keV with a fluence of 1 x 101” ions/cm’. Bovine aorta smooth muscle cells and Chinese hamster V79 cells were also capable of proliferating on ion-implanted SPU. These cells did not proliferate on the non-implanted SPU surface.

At this time, the mechanism for these remarkable improvements in cell adhesion and proliferation on an ion-implanted polymer surface is not clear. The present technique might be useful where controls of adhesion and proliferation of cells are required, such

Biomaterials 1993, Vol. 14 No. 12

960 Selective cell adhesion and proliferation: J.-S. Lee et a/.

as in developing hybrid artificial organs or functional devices (micro-machines and biosensors). Patterning of cells on ion-implanted domains may be obtained by using masks with different patterns or by controlling the direction of cell proliferation. This cell patterning may be applicable in various fields including neuro- bioscience or cell biology. For example, the patterning may be available for preparing a bio-device consisting of a junction of neuron cells and a field-effect transis- tor. Also, the patterning would be useful. for investigat- ing various cell behaviours including cell-to-cell communication, cell migration, cell adhesion and cell proliferation. A further study on the mechanism of cell adhesion and proliferation on ion-implanted polymer surfaces is now in progress.

A part of this work was performed under the Special Researchers’ Basic Science Program of RIKEN and STA. We wish to thank Mr Kazuaki Kira (Kanegafuchi

Chemical Industry) for providing segmented poly- urethane.

REFERENCES

Iwaki, M., Metal surface modi~~ation by ion implanta- tion. CRC Crit. Rev. Solid State Mater. Sci. 1989, 15, 473-508 Davenas, J., Thevenard, P., Boiteux, G., Fallavier, M. and Lu, X.L., Hydrogenated carbon layers produced by ion beam irradiation of PMMA and polystyrene films. Nucf. In&r. Methods 1990, B46, 317-323 Suzuki, Y., Kusakabe, M., Akiba, H., Kusakabe, K. and Iwaki, M., In vivo evaluation of antithrombogenicity for ion implanted silicone rubber using indium-Ill- tropolone platelets. Nucl. Instr. Methods 1991, B59/60, 698-704 Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minick, C.R., Culture of human endothelial cells derived from umbilical veins. J Cfin. invest. 1973, 52, 2745-2756 Schwartz, S.M., Selection and characterization of bovine aortic endothelial cell. In Vitro 1978, 14, 966- 980

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Biomaterials 1993, Vol. 14 No. 12