- Email: [email protected]
ppHEMA-modified silicone rubber film towards improving rabbit corneal epithelial cell attachment and growth
591
ppHEMA-modified silicone rubber film towards improving rabbit cornea1 epithelial cell attachment and growth Ging-Ho Hsiue* , Shyh-Dar Lee*, Chee-Chan Wang*, Michael Hum-1 Shiue* and Patricia Chuen-Tsuei Cham? *DepartKent bepartment
of Chemical Engineering, Nations/ Tsing Hua University, of Ophthalmology, Taichung Veterans General Hospital,
Hsinzhu, 300, Taiwan ROC; Taichung, 400, Taiwan ROC
A plasma polymerized HEMA (ppHEMA) film was prepared by plasma deposition polymerization onto an elastic material, silicone rubber. The surfaces of control, argon plasma-treated, and ppHEMA-modified silicone rubber were characterized by ESCA, FUR-ATR and SEM techniques. ESCA verified the respective chemical shift of control and ppHEMA-modified films. The presence of the ppHEMA was also verified by ESCA. The introduction of ppHEMA onto a hydrophobic support provided an adequate surface for rabbi cornea1 epithelial cell attachment and growth. Cell attachment and growth onto these surfaces were examined by light microscopy. Cell attachment onto the control and the argon plasma-treated surface was negligible, while improved attachment and growth of rabbi cornea1 epithelium cells was demonstrated on the ppHEMA-modified polymeric surface. The ppHEMA-modified silicone rubber surface demonstrated a confluent cell layer after 72 h. Keywords: Cell attachment, silicone rubber, HEMA, polymerization,
ppHEMA
Received 3 August 1992; revised 7 September 1992; accepted 8 December 1992
Plasma treatment and plasma deposition polymerization provide a unique and powerful method for the surface chemical modification of polymeric materials without altering their bulk properties*-3. These techniques offer possibilities to improve the performance of existing biomaterials and medical devices and for developing new biomaterials4-“. Surface modification through usage of plasma deposition offers several advantages, including ease of deposition on certain surfaces such as polytetrafluoroethylene, which are difficult to treat with conventional chemical methods. The resulting surfaces are also particle-free and sterile, due to the low amount of chemical introduced and the use of an ionizing atmosphere. The deposition films are thin, tightly adherent and can be deposited on complex geometries without affecting porosity or compliance7* ‘. A plasma deposition technique was mentioned by Gombotz and Hoffman9 in 1988, to modify the polymeric surfaces such as PE, PP and PET for providing biomaterial surfaces with hydrophilic properties. An ammonia gaseous plasma modification technique was used in 1990 by Sipehia et a1.l’ to modify polymeric surface, such as pHEMA-MMA copolymer, PMMA, PS and silicone Correspondence
to Dr G.-H. Hsiue.
8 1993 Buttenvorth-HeinemannLtd 0142-9612/93/060591-07
rubber membranes. The purpose of that research was to study enhancement of cell attachment and growth of rabbit cornea1 epithelial cells. An investigation into the ability of radiofrequency [RF) plasma surface depositions to improve endothelial cell growth in vitro was reported in 1990 by Ertel et al.“. Bovine aortic endothelium cell growth measured on films deposited from acetone, methanol and glutaraldehyde was correlated linearly with the oxygen content of the treated surfaces. A series of studies regarding surface modification of polymeric films with graft polymerization have been started by Wang and co-workers’2-‘4 using a plasma technique. The surface of silicone rubber was modified in the present study by plasma deposition polymerization of HEMA. These control, argon plasma-treated and ppHEMA-modified surfaces were used to assess the attachment and growth of rabbit cornea1 epthelium cells.
EXPERIMENTAL Materials The substrate polymerization
polymer used for plasma deposition was silicone rubber, MDX-4-4210 medical Biomaterials 1993, Vol. 14 No. 6
silicone rubber film: G.-H. Hsiue et al.
592
ppHEMA-modified
grade, purchased from Dow Corning (Midland, USA). The silicone rubber films were prepared by hot compression moulding (250 psi, 75% 30 min), The measured diameter was 9.5 mm and the thickness was 340pm. The culture medium was purchased from Boehringer Mannheim GmbH - Biochemica (Mannheim, Germany). Z-Hydroxylethyl methacrylate (HEMA) (MERCK) was redistilled under vacuum (bp, 54.O’C) before use. All other chemicals used in this study were of reagent grade and used as obtained.
with PBS, the epithelial layer was separated from the stroma and minced into small pieces with forceps and then placed in a tissue culture flask containing 3.5 ml of culture medium. The composition of the culture medium was 86 ml Minimum Essential Medium (MEM), 1 ml amino acid, 1 ml L-glutamine, 1 ml growth factor (EGF), 1 ml penicillin and 10 ml fetal bovine serum. The cultures were incubated in 5% CO,, 95% air at 37.6’C in a humidified incubator and changed every 48 h. The epithelial cell cultures became confluent after 7-10 d, after which cells were exposed to 1.0 ml of 0.1% trypsin in PBS, pH 7.3, for 2-3 min. The trypsin was aspirated and the cells dispersed and collected in 5.0 ml of culture medium. The cells in the medium were centrifuged and the pellet dispersed in 4.5 ml of culture medium for seeding onto the control, argon plasma-treated and pHEMA-grafted films. Throughout all the experiments, the unmodified silicone rubber film was used as the ‘control’ in the characterization and cell culture studies. The procedures for cell-seeding and the measurement of adhered cells onto control, argon plasma-treated and ppHEMAmodified surfaces were the same as described above for the plate culture. The number of cells seeded onto the samples was 2.4 X lo5 cells/cm’. After 72-96 h of incubation, the substrate materials were fixed with 1% formalin and stained with haematoxylin and eosin (HE) and observed under a light microscope.
Apparatus An FTIR-ATR spectrometer (BOMEM DA3.002) was used for characterizing the surfaces of the modified film and controls. A scanning electron microscope (HITACHI570) was used to analyse the morphology of the control and modified films. An ESCA-850 spectrometer manufactured by Shimadzu (Kyoto, Japan) was used to measure the control and ppHEMA-modified films at a pass energy of 1253.6 eV with an Mg Ka X-ray source. The ESCA data were processed with an ESPCA 210-S analyser.
Measurement of contact angle Static contact angles of water placed on plasma modified films were measured at 25OC and 65% relative humidity through use of the sessile drop method by using a drop of 2 ~1. The contact angle was read 1 min after the droplet was applied. Nine measurements on different surface places were also averaged. Deionized water was used for the measurements.
Radiofrequency
plasma treatment
The glow discharge reactor used was a Model PD-2 plasma deposition with a bell jar-type reaction cell, manufactured by Samco (Kyoto, Japan). The frequency applied was 13.56 MHz and an impedance matching unit was required. Plasma deposition pol~erization onto the polymeric film was carried out as follows. Polymeric films were placed over the electrode. The pressure in the bell jar was reduced to 0.05 torr, which was followed by the introduction of argon gas into the bell jar, and then evacuated to 0.05 torr. This process was repeated three times. Argon plasma was generated at 200 mtorr, 60 W and 60 s to pretreat the polymeric surface. The same process was then repeated three times through the introduction of a HEMA monomer vapour into the bell jar, Plasma was next generated at a given electric power and the films were exposed to plasma for a predetermined period of time. Several different powers and reaction time periods were used for the plasma deposition pol~erization. After the plasma treatment, argon gas was introduced into the bell jar reactor at a flow rate of 200 ml/min, for 20 min.
RESULTS AND DISCUSSION Modification of the substrate Measurement
of contact angle
The polymer films without plasma treatment exhibit a water contact angle of 105”, indicating that hydrophobicity of the polymer films is drastically decreased by plasma treatment (Figure 1). Plasma deposition polymerization of a water soluble monomer, HEMA, produces a permanently hyd~philic surface on the surface of hy~phobic polymer substrates which is entirely different from that being produced upon simple oxidation by plasma discharge. This is because the surface modified by the plasma deposition polymerization carries many macromolecular chains which are covalently bonded to the polymer substrate. The oxidized surface, meanwhile, only has oxygen-containing polar groups. The ppHEMA-modified films show a stable contact angle with time when being stored in deionized water. The ppHEMA-modified films which are stored in dry air have, however, revealed a gradual increase in contact angle over time (Figure 2). Therefore, in ppHEMA exposed to a hydrophilic envi~nment, OH groups are expressed at the surface at the ppHEMA-water interface while at the ppHEMA-air interface there is chain rotation to expose methyl groups at the surface’5*‘g.
ESCA study Cell culture Eyes were obtained from New Zealand white rabbits. The globes were washed with phosphate buffered saline (PBS), then transferred into a tissue culture flask containing PBS and the corneas were excised with ophthalmic scissors. The excised corneas were washed Biomaterials 1993, Vol. 14 No. 8
The polymeric surfaces of control, argon plasma-treated and ppHEMA-modified films were studied by measuring the core level spectra of Cls with ESCA. A strong peak at 285.75 eV, indicative of carbon-carbon bonds in control silicone rubber, was evident by the Cls spectra for the control silicone rubber surface (Figure 3a). The presence
ppHEMA-modified
silicone
rubber
Figure 3b. The Cls spectra exhibited a stronger intensity at a higher binding energy, indicating the formation of carbon-oxygen functionalities after plasma treatment. The high-binding energy region of the Cls spectra, as referenced by Clark et a1.17 and Dilks and Van Laeken”, could be fitted with three peaks which correspond to carbon atoms with a single bond to oxygen at 286.4 eV (e.g. alcohol, epoxy, ether, ester, hydroperoxy, peroxide], carbon atoms with two bonds to oxygen at 287.6 eV (e.g. aldehyde or ketone) and carbon atoms with three bonds to oxygen at 289.3 eV [e.g. carboxylic acid or esters). The
80 z
70
$ aI r, 6 5
593
film: G.-H. Hsiue et al.
60 50
-c-c-
J v6
40 30 20 10
0
0
Plasma
time
(s)
Figure 1 The effect of the plasma deposition time on the contact angle of silicone rubber film exposed to HEMA plasma (60 W, 0.1 torr).
80
'60
0
3
6
9
12
15
Storage
18
21
24
27
30
time Cd)
Figure2 Change of the water contact angle on standing in: 0, water; A, or air at room temperature for silicone rubber film modified with ppHEMA (60 W, 0.1 torr, 10 min). 285
290
of other peaks in Figure 3a was attributed to the absorbed or trapped oxygen on the silicone rubber surface. The ESCA Cls spectra for the silicone rubber film treated at an argon plasma, 60 W, 0.2 torr, 1 min are shown in
Eb(l eV/div)
Figure3 ESCA spectra of the silicone rubber film: a, control: b, argon plasma-treated (0.2 torr, 60 W, 60 s); c, ppHEMAmodified. Biomaterials
1993, Vol. 14 No. 8
594 Table 1
ppHEMA-modified Ratio of the corrected
peak area
Sample
Ols/Cls
C-H
c-o
c=o
o-c=0
Control-Sil PDP-Sil’
100 196
100 100
14.70 41.50
4.60 14.30
2.00 12.40
‘silicone treated by plasma deposition polymerization of HEMA.
Cls spectra of a ppHEMA-modified film are shown in Figure 3c, which verifies the presence of the modified ppHEMA. The relative intensity of the three components which have a high binding energy seemingly changes with the control and ppHEMA-modified films, as illustrated in Table 1. The Ols/Cls data of ppHEMA-modified films have, however, exhibited different values in comparison with those of control films. The relative amounts of the three components which have a higher binding energy are also different. The control film has a smaller amount (14.70) of low oxidized status than ppHEMA-modified silicone rubber has (41.50). It has exhibited a lower oxidized status surface for a ppHEMA-modified silicone rubber. A difference surface component is implied, by this significant difference, to be present on the modified film. This low oxidized component of the modified silicone rubber surface may possibly play a role in the initial attachment of cells coming from the culture medium onto the ppHEMA-modified surface.
silicone
rubber
The study of morphology polymer
film: G.-H. Hsiue et a/.
of plasma
deposition
Respective SEM photographs of control, argon plasmatreated and ppHEMA-modified silicone rubber films are presented in Figure 5. The surfaces of ppHEMAmodified films have been shown by a comparison between control (Figure Sa), argon plasma-treated (Figure 56) and ppHEMA-modified films (Figure SC) to be rougher than the surface of control substrates. The rough surface of a ppHEMA-modified silicone rubber is homogeneous.
Cell culture The attachment of rabbit epithelial cells onto the control, argon plasma-treated and ppHEMA-modified surfaces
FHR-ATR study The presence of ppHEMA has been confirmed by FTIRATR spectra as illustrated in Figure 4. Figure 4a represents the control silicone rubber film and Figure 4b represents ppHEMA-modified film. The absorption bands present at approximately 1720/cm are the characteristic absorption bands occurring for carbonyl groups of HEMA.
b
1725.6
3000
2000
Wavenumber
(cm
-1
)
Figure 4 FTIR-ATR spectra of: a, control silicone rubber film; b, ppHEMA-modified silicone rubber film treated by plasma deposition polymerization of HEMA. Biomaterials 1993, Vol.
14 No. 8
Figure5 SEM photographs (magnification X2000) of: a, control silicone rubber film; b, argon plasma-treated silicone rubber film; c, ppHEMA-modified silicone rubber film.
ppHEMA-modified
silicone rubber film: G.-H. Hsiue et al.
595
observed with a light microscope. A negligible amount of attachment of cells is present on the control and argon plasma-treated silicone rubber membranes (Figures 6 and 7). An improved attachment of epithelial cells towards the ppHEMA-modified silicone rubber membrane has, however, been observed, as illustrated in Figure 8. Cells attached to the ppHEMA-modified silicone rubber surface are significantly higher in
was
Figure 6 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to control silicone rubber film for 96 h.
Figure 8 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to ppHEMA-modified silicone rubber film at: a, 24 h; b, 48 h; c, 72 h.
Figure 7 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to argon plasma-treated silicone rubber film at: a, 24 h; b, 48 h.
number when compared with the control and argon plasma-treated surfaces. The toxicities of the control, argon plasma-treated and ppHEMA-modified silicone rubber membrane were assessed by a simple trypan blue exclusion test on the applied cells. These toxicity tests were negative. Approximately 99% of the cells were residual active cells. Photomicrographs of the rabbit cornea1 epithelial cell attached to control, argon plasma-treated and ppHEMAmodified silicone rubber surfaces stained with HE are shown in Figures 9, 10 and 11, respectively. The ppHEMA-modified silicone rubber surface has been confluenced after seeding the cells for 72 h. Biomaterials 1993. Vol. 14 No. 8
596
ppHEMA-modified
silicone rubber film: G.-H. Hsiue et a/.
The introduction of support has provided cornea1 epithelial cell two hours after seeding silicone rubber surface
ppHEMA onto a hydrophobic an adequate surface for rabbit attachment and growth. Seventythe cells, the ppHEMA-modified was confluenced.
ACKNOWLEDGEMENTS Financial support of this work by the National Science Council of the Republic of China (NCS-81-0412-B-075A510) is gratefully acknowledged. The authors would also like to thank the Research Laboratory of Susumu Industrial Co. Ltd, Kyoto, Japan, for the use of its ESCA instrument. Figure9 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to control silicone rubber film, stained with HE.
REFERENCES 1
2
3
4
5
6 Figure10 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to argon plasma-treated (60 W, 0.2 torr, 60 s) silicone rubber film, stained with HE.
7 6
9
10
11
Figure11 Photomicrograph (original magnification X250) of rabbit cornea1 epithelial cell attached to ppHEMA-modified silicone rubber film stained with HE.
CONCLUSION ppHEMA has been incorporated onto a polymer surface through the use of plasma deposition polymerization. Biomaterials
1993, Vol. 14 No. 8
12
13
14
Boenig, H.V., Fundamentals of Plasma Chemistry and Technology, Technomic Publishing Co., Lancaster, Basel, Switzerland, 1988 Yasuda, H., Plasma Polymerization, Academic Press, NY, USA, 1965 Yasuda, H.K. (Ed.), Polymerization and plasma interactions with polymeric materials, 1. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 36-50 Hoffman, AS., Modification of material surfaces to affect how they interact with blood, Ann. NY Acad. Sci. 1987, 146, 96-101 Hoffman, A.S., Biomedical applications of plasma gas discharge processes, J. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 341-359 Hoffman, AS., Adsorption and immobilization of protein on gas discharge treated surface, J. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 341-359 Yasuda, H., Glow discharge polymerization,]. Polym. Sci. 1981, 16,199-293 Hollahan, J.R., Wydeven, T. and Johnson, C.C., Combination moisture resistant and anti-reflection plasma polymerized thin films for optical coatings, Appl. Opt. 1979, 13,1844-1848 Gombotz, W.R. and Hoffman, AS., Functionalization of polymeric films by plasma polymerization of ally1 alcohol and ally1 amine, 1. Appl. Polym. Sci., Appl. Polym. Symp. 1986, 42, 285-303 Sipehia, R., Garfinkle, A., Jackson, W.B. and Chang, T.M.S., Towards an artificial cornea: surface modification of optically clear, oxygen permeable soft contact lens material by ammonia plasma modification technique for the enhanced attachment and growth of cornea1 epithelial cells, Biomater., Artif. Cells, Artif. Organs 1990, 16, 643-655 Ertel, S.I., Ratner, B.D. and Horbett, T.S., Radiofrequency plasma deposition of oxygen-containing films on polystyrene and poly(ethylene terephthalate) substrates improves endothelial cell growth, 1. Biomed. Mater. Res. 1990, 24,1637-1659 Wang, CC. and Hsiue, G.H., Oxidation of polyethylene surface by glow discharge and subsequent graft copolymerization of acrylic acid, I. Polym. Sci., Polym. Chem. Ed. [in press] Wang, CC. and Hsiue, G.H., Immobilization of glucose oxidase on polyethylene film using a plasma induced graft copolymerization process, 1. Biomater. Sci., Polym. Ed. (in press) Hsiue, G.H., Lee, S.D., Wang, CC. and Chang, Patricia
ppHEMA-modified
597
silicone rubber film: G.-H. Hsiue et al.
CT., The effect of plasma induced graft copolymerization of HEMA on silicone rubber towards improving cornea1 epithelial cells growth, I. Eiomater. Sci., Polym. Ed. [in press] Holly, F.J. and Refojo, F.M., in Hydrogels for Medical and Related Applications (Ed. J.D. Andrade) ACS Symposium Series, Vol. 31, American Chemical Society, Washington, DC, USA, 1976, p 252 Morra, M., Occhiello, E. and Garbassi, F., On the
17
18
wettabili~ of poly(2-hyd~xyethylmethac~late), j. Coi~o~d Interface Sci. 1992, 149,84-91 Clark, T., Cromarty, B.J. and Dilks, A.H.R., Application of ESCA to polymer chemistry. XVII. Systematic investigation of the core levels of simple homopolymers, 1. Pofym. Sci., Polym. Chem. Ed. 1978, 16, 791-820 Dilks, A. and Laeken, A. Van, Phys~cochemjcal Aspects of Polymer Surface (Ed. K.L. Mittal), Vol. 1, Plenum Press, NY, USA, 1983, pp 749-772
One-day Workshop
Progress in Biomaterials
Surfaces
CSMA Ltd, Armstrong House, Manchester, UK 20th October 1993 The workshop will combine lecture presen~tions with practicaf demonstrations of exp~rimen~l techniques. There will also be a Consultancy session during which delegates may discuss, In confidence, any matters which may be of particular interest to them.
Topics 0 Surface energy and its ~urement 0 Surface plasmon resonance
0 XPS and ToFSIMS 0 Surface phenomena in medicine and dentistry
Speakers 0 Dr J.Davies (Kodak Clinical Diagnostics) 0 Dr J.Nicholson (LGC & King’s College London) W Dr D.Watts (Manchester Dental School)
0 Dr R.West (CSMA Ltd)
For further information please contact: Samantha Chadwick, CSMA Ltd, Armstrong House, Oxford Road, Manchester, Ml 7ED, UK. Organized by the Laboratory of the Government
Chemist and CSNA
Ltd.
Biomaterials
1993, Vol.
14 No. 8
ppHEMA-modified silicone rubber film towards improving rabbit cornea1 epithelial cell attachment and growth Ging-Ho Hsiue* , Shyh-Dar Lee*, Chee-Chan Wang*, Michael Hum-1 Shiue* and Patricia Chuen-Tsuei Cham? *DepartKent bepartment
of Chemical Engineering, Nations/ Tsing Hua University, of Ophthalmology, Taichung Veterans General Hospital,
Hsinzhu, 300, Taiwan ROC; Taichung, 400, Taiwan ROC
A plasma polymerized HEMA (ppHEMA) film was prepared by plasma deposition polymerization onto an elastic material, silicone rubber. The surfaces of control, argon plasma-treated, and ppHEMA-modified silicone rubber were characterized by ESCA, FUR-ATR and SEM techniques. ESCA verified the respective chemical shift of control and ppHEMA-modified films. The presence of the ppHEMA was also verified by ESCA. The introduction of ppHEMA onto a hydrophobic support provided an adequate surface for rabbi cornea1 epithelial cell attachment and growth. Cell attachment and growth onto these surfaces were examined by light microscopy. Cell attachment onto the control and the argon plasma-treated surface was negligible, while improved attachment and growth of rabbi cornea1 epithelium cells was demonstrated on the ppHEMA-modified polymeric surface. The ppHEMA-modified silicone rubber surface demonstrated a confluent cell layer after 72 h. Keywords: Cell attachment, silicone rubber, HEMA, polymerization,
ppHEMA
Received 3 August 1992; revised 7 September 1992; accepted 8 December 1992
Plasma treatment and plasma deposition polymerization provide a unique and powerful method for the surface chemical modification of polymeric materials without altering their bulk properties*-3. These techniques offer possibilities to improve the performance of existing biomaterials and medical devices and for developing new biomaterials4-“. Surface modification through usage of plasma deposition offers several advantages, including ease of deposition on certain surfaces such as polytetrafluoroethylene, which are difficult to treat with conventional chemical methods. The resulting surfaces are also particle-free and sterile, due to the low amount of chemical introduced and the use of an ionizing atmosphere. The deposition films are thin, tightly adherent and can be deposited on complex geometries without affecting porosity or compliance7* ‘. A plasma deposition technique was mentioned by Gombotz and Hoffman9 in 1988, to modify the polymeric surfaces such as PE, PP and PET for providing biomaterial surfaces with hydrophilic properties. An ammonia gaseous plasma modification technique was used in 1990 by Sipehia et a1.l’ to modify polymeric surface, such as pHEMA-MMA copolymer, PMMA, PS and silicone Correspondence
to Dr G.-H. Hsiue.
8 1993 Buttenvorth-HeinemannLtd 0142-9612/93/060591-07
rubber membranes. The purpose of that research was to study enhancement of cell attachment and growth of rabbit cornea1 epithelial cells. An investigation into the ability of radiofrequency [RF) plasma surface depositions to improve endothelial cell growth in vitro was reported in 1990 by Ertel et al.“. Bovine aortic endothelium cell growth measured on films deposited from acetone, methanol and glutaraldehyde was correlated linearly with the oxygen content of the treated surfaces. A series of studies regarding surface modification of polymeric films with graft polymerization have been started by Wang and co-workers’2-‘4 using a plasma technique. The surface of silicone rubber was modified in the present study by plasma deposition polymerization of HEMA. These control, argon plasma-treated and ppHEMA-modified surfaces were used to assess the attachment and growth of rabbit cornea1 epthelium cells.
EXPERIMENTAL Materials The substrate polymerization
polymer used for plasma deposition was silicone rubber, MDX-4-4210 medical Biomaterials 1993, Vol. 14 No. 6
silicone rubber film: G.-H. Hsiue et al.
592
ppHEMA-modified
grade, purchased from Dow Corning (Midland, USA). The silicone rubber films were prepared by hot compression moulding (250 psi, 75% 30 min), The measured diameter was 9.5 mm and the thickness was 340pm. The culture medium was purchased from Boehringer Mannheim GmbH - Biochemica (Mannheim, Germany). Z-Hydroxylethyl methacrylate (HEMA) (MERCK) was redistilled under vacuum (bp, 54.O’C) before use. All other chemicals used in this study were of reagent grade and used as obtained.
with PBS, the epithelial layer was separated from the stroma and minced into small pieces with forceps and then placed in a tissue culture flask containing 3.5 ml of culture medium. The composition of the culture medium was 86 ml Minimum Essential Medium (MEM), 1 ml amino acid, 1 ml L-glutamine, 1 ml growth factor (EGF), 1 ml penicillin and 10 ml fetal bovine serum. The cultures were incubated in 5% CO,, 95% air at 37.6’C in a humidified incubator and changed every 48 h. The epithelial cell cultures became confluent after 7-10 d, after which cells were exposed to 1.0 ml of 0.1% trypsin in PBS, pH 7.3, for 2-3 min. The trypsin was aspirated and the cells dispersed and collected in 5.0 ml of culture medium. The cells in the medium were centrifuged and the pellet dispersed in 4.5 ml of culture medium for seeding onto the control, argon plasma-treated and pHEMA-grafted films. Throughout all the experiments, the unmodified silicone rubber film was used as the ‘control’ in the characterization and cell culture studies. The procedures for cell-seeding and the measurement of adhered cells onto control, argon plasma-treated and ppHEMAmodified surfaces were the same as described above for the plate culture. The number of cells seeded onto the samples was 2.4 X lo5 cells/cm’. After 72-96 h of incubation, the substrate materials were fixed with 1% formalin and stained with haematoxylin and eosin (HE) and observed under a light microscope.
Apparatus An FTIR-ATR spectrometer (BOMEM DA3.002) was used for characterizing the surfaces of the modified film and controls. A scanning electron microscope (HITACHI570) was used to analyse the morphology of the control and modified films. An ESCA-850 spectrometer manufactured by Shimadzu (Kyoto, Japan) was used to measure the control and ppHEMA-modified films at a pass energy of 1253.6 eV with an Mg Ka X-ray source. The ESCA data were processed with an ESPCA 210-S analyser.
Measurement of contact angle Static contact angles of water placed on plasma modified films were measured at 25OC and 65% relative humidity through use of the sessile drop method by using a drop of 2 ~1. The contact angle was read 1 min after the droplet was applied. Nine measurements on different surface places were also averaged. Deionized water was used for the measurements.
Radiofrequency
plasma treatment
The glow discharge reactor used was a Model PD-2 plasma deposition with a bell jar-type reaction cell, manufactured by Samco (Kyoto, Japan). The frequency applied was 13.56 MHz and an impedance matching unit was required. Plasma deposition pol~erization onto the polymeric film was carried out as follows. Polymeric films were placed over the electrode. The pressure in the bell jar was reduced to 0.05 torr, which was followed by the introduction of argon gas into the bell jar, and then evacuated to 0.05 torr. This process was repeated three times. Argon plasma was generated at 200 mtorr, 60 W and 60 s to pretreat the polymeric surface. The same process was then repeated three times through the introduction of a HEMA monomer vapour into the bell jar, Plasma was next generated at a given electric power and the films were exposed to plasma for a predetermined period of time. Several different powers and reaction time periods were used for the plasma deposition pol~erization. After the plasma treatment, argon gas was introduced into the bell jar reactor at a flow rate of 200 ml/min, for 20 min.
RESULTS AND DISCUSSION Modification of the substrate Measurement
of contact angle
The polymer films without plasma treatment exhibit a water contact angle of 105”, indicating that hydrophobicity of the polymer films is drastically decreased by plasma treatment (Figure 1). Plasma deposition polymerization of a water soluble monomer, HEMA, produces a permanently hyd~philic surface on the surface of hy~phobic polymer substrates which is entirely different from that being produced upon simple oxidation by plasma discharge. This is because the surface modified by the plasma deposition polymerization carries many macromolecular chains which are covalently bonded to the polymer substrate. The oxidized surface, meanwhile, only has oxygen-containing polar groups. The ppHEMA-modified films show a stable contact angle with time when being stored in deionized water. The ppHEMA-modified films which are stored in dry air have, however, revealed a gradual increase in contact angle over time (Figure 2). Therefore, in ppHEMA exposed to a hydrophilic envi~nment, OH groups are expressed at the surface at the ppHEMA-water interface while at the ppHEMA-air interface there is chain rotation to expose methyl groups at the surface’5*‘g.
ESCA study Cell culture Eyes were obtained from New Zealand white rabbits. The globes were washed with phosphate buffered saline (PBS), then transferred into a tissue culture flask containing PBS and the corneas were excised with ophthalmic scissors. The excised corneas were washed Biomaterials 1993, Vol. 14 No. 8
The polymeric surfaces of control, argon plasma-treated and ppHEMA-modified films were studied by measuring the core level spectra of Cls with ESCA. A strong peak at 285.75 eV, indicative of carbon-carbon bonds in control silicone rubber, was evident by the Cls spectra for the control silicone rubber surface (Figure 3a). The presence
ppHEMA-modified
silicone
rubber
Figure 3b. The Cls spectra exhibited a stronger intensity at a higher binding energy, indicating the formation of carbon-oxygen functionalities after plasma treatment. The high-binding energy region of the Cls spectra, as referenced by Clark et a1.17 and Dilks and Van Laeken”, could be fitted with three peaks which correspond to carbon atoms with a single bond to oxygen at 286.4 eV (e.g. alcohol, epoxy, ether, ester, hydroperoxy, peroxide], carbon atoms with two bonds to oxygen at 287.6 eV (e.g. aldehyde or ketone) and carbon atoms with three bonds to oxygen at 289.3 eV [e.g. carboxylic acid or esters). The
80 z
70
$ aI r, 6 5
593
film: G.-H. Hsiue et al.
60 50
-c-c-
J v6
40 30 20 10
0
0
Plasma
time
(s)
Figure 1 The effect of the plasma deposition time on the contact angle of silicone rubber film exposed to HEMA plasma (60 W, 0.1 torr).
80
'60
0
3
6
9
12
15
Storage
18
21
24
27
30
time Cd)
Figure2 Change of the water contact angle on standing in: 0, water; A, or air at room temperature for silicone rubber film modified with ppHEMA (60 W, 0.1 torr, 10 min). 285
290
of other peaks in Figure 3a was attributed to the absorbed or trapped oxygen on the silicone rubber surface. The ESCA Cls spectra for the silicone rubber film treated at an argon plasma, 60 W, 0.2 torr, 1 min are shown in
Eb(l eV/div)
Figure3 ESCA spectra of the silicone rubber film: a, control: b, argon plasma-treated (0.2 torr, 60 W, 60 s); c, ppHEMAmodified. Biomaterials
1993, Vol. 14 No. 8
594 Table 1
ppHEMA-modified Ratio of the corrected
peak area
Sample
Ols/Cls
C-H
c-o
c=o
o-c=0
Control-Sil PDP-Sil’
100 196
100 100
14.70 41.50
4.60 14.30
2.00 12.40
‘silicone treated by plasma deposition polymerization of HEMA.
Cls spectra of a ppHEMA-modified film are shown in Figure 3c, which verifies the presence of the modified ppHEMA. The relative intensity of the three components which have a high binding energy seemingly changes with the control and ppHEMA-modified films, as illustrated in Table 1. The Ols/Cls data of ppHEMA-modified films have, however, exhibited different values in comparison with those of control films. The relative amounts of the three components which have a higher binding energy are also different. The control film has a smaller amount (14.70) of low oxidized status than ppHEMA-modified silicone rubber has (41.50). It has exhibited a lower oxidized status surface for a ppHEMA-modified silicone rubber. A difference surface component is implied, by this significant difference, to be present on the modified film. This low oxidized component of the modified silicone rubber surface may possibly play a role in the initial attachment of cells coming from the culture medium onto the ppHEMA-modified surface.
silicone
rubber
The study of morphology polymer
film: G.-H. Hsiue et a/.
of plasma
deposition
Respective SEM photographs of control, argon plasmatreated and ppHEMA-modified silicone rubber films are presented in Figure 5. The surfaces of ppHEMAmodified films have been shown by a comparison between control (Figure Sa), argon plasma-treated (Figure 56) and ppHEMA-modified films (Figure SC) to be rougher than the surface of control substrates. The rough surface of a ppHEMA-modified silicone rubber is homogeneous.
Cell culture The attachment of rabbit epithelial cells onto the control, argon plasma-treated and ppHEMA-modified surfaces
FHR-ATR study The presence of ppHEMA has been confirmed by FTIRATR spectra as illustrated in Figure 4. Figure 4a represents the control silicone rubber film and Figure 4b represents ppHEMA-modified film. The absorption bands present at approximately 1720/cm are the characteristic absorption bands occurring for carbonyl groups of HEMA.
b
1725.6
3000
2000
Wavenumber
(cm
-1
)
Figure 4 FTIR-ATR spectra of: a, control silicone rubber film; b, ppHEMA-modified silicone rubber film treated by plasma deposition polymerization of HEMA. Biomaterials 1993, Vol.
14 No. 8
Figure5 SEM photographs (magnification X2000) of: a, control silicone rubber film; b, argon plasma-treated silicone rubber film; c, ppHEMA-modified silicone rubber film.
ppHEMA-modified
silicone rubber film: G.-H. Hsiue et al.
595
observed with a light microscope. A negligible amount of attachment of cells is present on the control and argon plasma-treated silicone rubber membranes (Figures 6 and 7). An improved attachment of epithelial cells towards the ppHEMA-modified silicone rubber membrane has, however, been observed, as illustrated in Figure 8. Cells attached to the ppHEMA-modified silicone rubber surface are significantly higher in
was
Figure 6 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to control silicone rubber film for 96 h.
Figure 8 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to ppHEMA-modified silicone rubber film at: a, 24 h; b, 48 h; c, 72 h.
Figure 7 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to argon plasma-treated silicone rubber film at: a, 24 h; b, 48 h.
number when compared with the control and argon plasma-treated surfaces. The toxicities of the control, argon plasma-treated and ppHEMA-modified silicone rubber membrane were assessed by a simple trypan blue exclusion test on the applied cells. These toxicity tests were negative. Approximately 99% of the cells were residual active cells. Photomicrographs of the rabbit cornea1 epithelial cell attached to control, argon plasma-treated and ppHEMAmodified silicone rubber surfaces stained with HE are shown in Figures 9, 10 and 11, respectively. The ppHEMA-modified silicone rubber surface has been confluenced after seeding the cells for 72 h. Biomaterials 1993. Vol. 14 No. 8
596
ppHEMA-modified
silicone rubber film: G.-H. Hsiue et a/.
The introduction of support has provided cornea1 epithelial cell two hours after seeding silicone rubber surface
ppHEMA onto a hydrophobic an adequate surface for rabbit attachment and growth. Seventythe cells, the ppHEMA-modified was confluenced.
ACKNOWLEDGEMENTS Financial support of this work by the National Science Council of the Republic of China (NCS-81-0412-B-075A510) is gratefully acknowledged. The authors would also like to thank the Research Laboratory of Susumu Industrial Co. Ltd, Kyoto, Japan, for the use of its ESCA instrument. Figure9 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to control silicone rubber film, stained with HE.
REFERENCES 1
2
3
4
5
6 Figure10 Photomicrograph (original magnification X125) of rabbit cornea1 epithelial cell attached to argon plasma-treated (60 W, 0.2 torr, 60 s) silicone rubber film, stained with HE.
7 6
9
10
11
Figure11 Photomicrograph (original magnification X250) of rabbit cornea1 epithelial cell attached to ppHEMA-modified silicone rubber film stained with HE.
CONCLUSION ppHEMA has been incorporated onto a polymer surface through the use of plasma deposition polymerization. Biomaterials
1993, Vol. 14 No. 8
12
13
14
Boenig, H.V., Fundamentals of Plasma Chemistry and Technology, Technomic Publishing Co., Lancaster, Basel, Switzerland, 1988 Yasuda, H., Plasma Polymerization, Academic Press, NY, USA, 1965 Yasuda, H.K. (Ed.), Polymerization and plasma interactions with polymeric materials, 1. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 36-50 Hoffman, AS., Modification of material surfaces to affect how they interact with blood, Ann. NY Acad. Sci. 1987, 146, 96-101 Hoffman, A.S., Biomedical applications of plasma gas discharge processes, J. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 341-359 Hoffman, AS., Adsorption and immobilization of protein on gas discharge treated surface, J. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 341-359 Yasuda, H., Glow discharge polymerization,]. Polym. Sci. 1981, 16,199-293 Hollahan, J.R., Wydeven, T. and Johnson, C.C., Combination moisture resistant and anti-reflection plasma polymerized thin films for optical coatings, Appl. Opt. 1979, 13,1844-1848 Gombotz, W.R. and Hoffman, AS., Functionalization of polymeric films by plasma polymerization of ally1 alcohol and ally1 amine, 1. Appl. Polym. Sci., Appl. Polym. Symp. 1986, 42, 285-303 Sipehia, R., Garfinkle, A., Jackson, W.B. and Chang, T.M.S., Towards an artificial cornea: surface modification of optically clear, oxygen permeable soft contact lens material by ammonia plasma modification technique for the enhanced attachment and growth of cornea1 epithelial cells, Biomater., Artif. Cells, Artif. Organs 1990, 16, 643-655 Ertel, S.I., Ratner, B.D. and Horbett, T.S., Radiofrequency plasma deposition of oxygen-containing films on polystyrene and poly(ethylene terephthalate) substrates improves endothelial cell growth, 1. Biomed. Mater. Res. 1990, 24,1637-1659 Wang, CC. and Hsiue, G.H., Oxidation of polyethylene surface by glow discharge and subsequent graft copolymerization of acrylic acid, I. Polym. Sci., Polym. Chem. Ed. [in press] Wang, CC. and Hsiue, G.H., Immobilization of glucose oxidase on polyethylene film using a plasma induced graft copolymerization process, 1. Biomater. Sci., Polym. Ed. (in press) Hsiue, G.H., Lee, S.D., Wang, CC. and Chang, Patricia
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CT., The effect of plasma induced graft copolymerization of HEMA on silicone rubber towards improving cornea1 epithelial cells growth, I. Eiomater. Sci., Polym. Ed. [in press] Holly, F.J. and Refojo, F.M., in Hydrogels for Medical and Related Applications (Ed. J.D. Andrade) ACS Symposium Series, Vol. 31, American Chemical Society, Washington, DC, USA, 1976, p 252 Morra, M., Occhiello, E. and Garbassi, F., On the
17
18
wettabili~ of poly(2-hyd~xyethylmethac~late), j. Coi~o~d Interface Sci. 1992, 149,84-91 Clark, T., Cromarty, B.J. and Dilks, A.H.R., Application of ESCA to polymer chemistry. XVII. Systematic investigation of the core levels of simple homopolymers, 1. Pofym. Sci., Polym. Chem. Ed. 1978, 16, 791-820 Dilks, A. and Laeken, A. Van, Phys~cochemjcal Aspects of Polymer Surface (Ed. K.L. Mittal), Vol. 1, Plenum Press, NY, USA, 1983, pp 749-772
One-day Workshop
Progress in Biomaterials
Surfaces
CSMA Ltd, Armstrong House, Manchester, UK 20th October 1993 The workshop will combine lecture presen~tions with practicaf demonstrations of exp~rimen~l techniques. There will also be a Consultancy session during which delegates may discuss, In confidence, any matters which may be of particular interest to them.
Topics 0 Surface energy and its ~urement 0 Surface plasmon resonance
0 XPS and ToFSIMS 0 Surface phenomena in medicine and dentistry
Speakers 0 Dr J.Davies (Kodak Clinical Diagnostics) 0 Dr J.Nicholson (LGC & King’s College London) W Dr D.Watts (Manchester Dental School)
0 Dr R.West (CSMA Ltd)
For further information please contact: Samantha Chadwick, CSMA Ltd, Armstrong House, Oxford Road, Manchester, Ml 7ED, UK. Organized by the Laboratory of the Government
Chemist and CSNA
Ltd.
Biomaterials
1993, Vol.
14 No. 8