artificial cornea: surface modification of silicone …rdconner/536/additional/artificial cornea...

Biomaterids 17 (1996) 567-595

0 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved

0142-9612/96/$15.00

Artificial cornea: surface modification of silicone rubber membrane by graft polymerization of pHEMA via glow discharge

Shyh-Dar Lee*, Ging-Ho Hsiue” , Chen-Yu Kao* and Patricia Chuen-Tsuei Changt *Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC; and tDepartment of Ophthalmology, Taichung Veterans General Hospital, Taichung, Taiwan, ROC

A method for producing various surfaces of silicone rubber membrane (SR) was developed in this study

by grafting various amounts of poly(Bhydroxy ethyl methacrylate) (pHEMA) onto SR by plasma-induced

grafted polymerization (PIP) as a homobifunctional membrane. The elemental composition and different

carbon bindings on the surface of SR were examined by electron spectroscopy for chemical analysis with

the amount of OlslCls being approximately 0.7 at 1 min, 60 W, 200 mTorr of Ar-plasma treatment. The

peroxide group introduced on SR was measured via l,l-diphenyl-2-picrylhydrazyl (DPPH) and the

amount of 6.85 x lo-* mol cm-’ reached optimum value at 1 min of Ar-plasma treatment. After Ar-plasma

treated SR, the peroxide group (33D peak) was introduced on the surface of SR by negative spectra of

secondary ion mass spectroscopy analysis, whereas ester groups (72D peak) were observed for pHEMA-

grafted SR. For the in vitro test, the influence of various surfaces of SR on attachment and growth of rabbit

cornea1 epithelial cells (CEC) was studied by cell culture assay. These results indicated that 56-

150pgcm-’ of pHEMA grafted onto SR were suitable values for attachment and growth of CEC. On the

contrary, the large grafted amounts (500-165Opg cm-‘) of pHEMA on SR were insufficient for attachment

and growth of CEC. For the in viva test, the migration of CEC from host cornea to implant was investigated

by slit lamp microscopy. The experimental results indicated that SRs grafted with pHEMA were

completely covered with CEC 3 weeks after implantation of the membranes into the host cornea. These

results provide a valuable reference for developing an artificial cornea.

Keywords: Poly(2-hydroxyethyl methacrylate), silicone rubber membrane, plasma-induced grafted

polymerization, homobifunctional membrane, cornea/ epithelial cell, penetrating keratoplasty

Received 18 January 1995; accepted 1 June 1995

When progressive cornea1 ulceration or penetrating cornea1 injury with tissue loss occurs, emergency penetrating keratoplasty (PK) or lamella path graft must be performed to restore ocular integrity and avoid further complications, such as extensive chamber angle synechia, angle closure glaucoma and endophthalmitis. A plastic fibre meshwork supporting plate, i.e. Cardona implant, was first studied for keratoprosthesis’,‘. The generation of keratoprosthesis is Girard keratoprosthesis based on modification of the original Cardona implant3. Girard keratoprosthesis consists primarily of implanting the keratoprosthesis with the skirt on the surface of the cornea. In 1991, Kirkham and Dangle4 reported that coating poly(methy1 methacrylate) (PhJMA) intracorneal keratoprosthesis with type I collagen would enhance the biocompatibility of an implant. The aforementioned

Correspondence to Dr G.-H. Hsiue.

keratoprosthesis implants are restricted in clinical applications owing to the fact that the size of prosthesis implant is not adjustable and the position of surgery must be in the ocular centre. In 1991, Kobayashi et ~1.~ reported that a polyvinyl chloride hydrogel immobilized with collagen in a contact lens shape was used for keratoprosthesis. In their investigation, a lamellar keratoplasty technique was used and the implant was rejected i?om the host cornea one month after implantation.

The preparation of an artificial cornea has been under study in our laboratory for quite some time6. The results obtained so far indicate that three issues must be dealt with in order for an artificial cornea to be obtained. First, the implant must be completely covered with the cornea1 epithelial cells (CEC). Second, downgrowtb of CEC has to be suppressed when the implant is maintained in the living cornea for a long period of time. Finally, the process of

587 Biomaterials 1996, Vol. 17 No. 6

588 Formation of artificial cornea from pHEMA-grafted silicone rubber membranes: S.-D. Lee et al.

wound healing must be considered because the implant needs to be tightly fixed on the host cornea. Based on the above information, material properties such as hydrophilicity/hydrophobicity, softness/hardness, tensile strength and biocompatibility are considered in preparing an artificial cornea.

The hydrogel of poly(2-hydroxy ethyl methacrylate) (pHEMA) is an inhibitor for cell attachment and exhibits good biocompatibility with the tissue. Wichterle and Lim made the first applications of synthetic hydrogels based on pHEMA7. However, poor mechanical properties were obtained for the hydrogel and the region for biomedical application was restricted.

Surface modifications of a polymer have been made by various methods, such as oxidation by flame or chemical reagent*, corona dischargeg, UV irradiationlO, ozonell, or gamma ray irradiation1”r3. The primary advantage of plasma-induced grafted polymerization is that the location for grafting polymer is limited at the surface region of the polymer material without altering the bulk properties14. The good mechanical properties and relatively low cost for conventional plastic and elastomer materials was utilized”. In addition, most of the surfaces of the elastomer were hydrophobic, Furthermore, the hydrophobicity could cause poor affinity of cell attachment.

In this study, a highly biocompatible polymer membrane is prepared by surface modification in an attempt to develop further a useful cornea for clinical applications. A homobifunctional membrane is also prepared. For the homobifunctional membrane, pHEMA is grafted onto both sides of silicone rubber (SR) 16,17. The grafting of pHEMA improves the attachment and growth of CEC. Additionally, surface characterizations of membranes are carried out using attenuated total reflection (ATR), electron spectroscopy for chemical analysis (ESCA), secondary ion mass spectroscopy (SIMS), scanning electron microscopy (SEM) and a contact angle meter. The toxicity and biocompatibility of membranes have also been investigated via in vitro and in vivo tests.

EXPERIMENTAL DETAILS

Materials

Silicone rubber membrane (MDX4-4210, medical grade elastomers) was purchased from Dow Corning Corporation (Midland, MI, USA). The SR thickness of 340-360 pm was achieved by hot compression moulding (25Opsig, 75°C) and the area was 1 cm’. The HEMA monomer, as supplied by Merck Chemical Company Inc. (Darmstadt, Germany), was redistilled under vacuum (1-2 Torr, 54°C) so as to free it from the inhibitor. All other solutions used were of reagents of analytical grade.

Plasma treatment

The detailed procedure has been described in a previous work”. SR was used and activated by Ar- plasma treatment. Moreover, the peroxide group was produced by exposing samples in oxygen gas.

Peroxide determination

Plasma-treated membranes were put into a degassed benzene solution of l,l-diphenyl-2picrylhydrazyl (DPPH) and maintained at 80°C for 24 h to decompose the formation of peroxides on and near the membrane surfaces, in accordance with earlier reports”. The DPPH molecules consumed were measured according to the difference in transmittance at 520nm between the control and Ar-plasma-treated membranes. The absorption coefficient of DPPH at 520 nm is 1.18 x lo4

1 mol-’ cm-l. ,

ATR-FTIR

KRS-5 (Harrick) was used for the crystalline cell of ATR-FTIR (Bomem DA 3.002). Scanning was carried out from 4000 cm-’ (2.5pm) to 650cm-’ (15.4pm) to confirm the formation of grafted polymerization.

ESCA

An ESCA Perkin-Elmer, PHI 1600 spectrometer was utilized. ESCA data analysis was processed with PHI- matlad. The analysis current was 250 W, and a take- off angle of 60” was used to carry out the ESCA measurement of various silicone rubber membranes at a pass energy of 1253.6eV with an Mg Kcc X-ray source.

SIMS

The mass spectra of silicone rubber membrane before and after plasma treatment were measured by SIMS (Cameca, ims-4f). The primary Ar+ ion beam was accelerated with a voltage of 3 kV and its beam current was 50mA. Such a low current is effective in preventing any charge of the sample surfaces. Moreover, the samples were covered by an electron shower to compensate for the charge-up. During measurements, the pressure was maintained at 3 x lo-’ Torr. Also, Cameca, ims-4f was used.

Contact angle analysis

Static contact angles of water on plasma-treated and pHEMA-grafted SRs were maintained by a relative humidity meter (Today) in an air-conditioned room. The SRs were measured at 25°C and 65% relative humidity (RH) with the sessile drop methodlg (Emra model G-l). A 2 ~1 deionized water bubble was placed onto the surface of SR using a micropipette. Ten drops were recorded and averaged for each membrane.

In vitro test

Cell culture assay6 Eyes were obtained from New Zealand white rabbits. The globes were washed with sterilized phosphate- buffered salines (PBS), then transferred into a tissue culture flask containing PBS. Next, the corneas were excised with ophthalmic scissors. The excised corneas were washed 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 culture medium. The composition of culture medium was 86ml of Eagle’s

Biomaterials 1996, Vol. 17 No. 6

Formation of artificial cornea from pHEMA-grafted silicone rubber membranes: S.-D. Lee et al. 589

minimum essential medium (EMEM), 1 ml non- essential amino acid, 1 ml L-glutamine, 1 ml peni- cillin-streptomycin (10 000 units ml-‘), 1 ml epidermal growth factor and lOm1 fetal calf serum. All of the cell culture components were purchased from Gibco BRL Life Technologies, Inc, Tokyo, Japan. The cell cultures were incubated in 5% COz/air at 37.6”C in a humidified incubator; in addition, the cell medium was changed every 48 h. The epithelial cell became confluent after 7-10d, then disaggregated with 0.25% trypsin to

,perform the next cell culture experiment.

Cell attachment and growth assay The epithelial cells were attached to the modified SR surfaces. The modified membranes were sterilized by autoclave (121psi, 120°C) and then placed on the 24- well tissue culture plate (Linbro, McLean, Virginia, USA). The numbers of epithelial cells attached onto various SR surfaces were measured on the 8th hour after epithelial cells were seeded onto the surfaces at a density of lo5 cells well-‘. The CEC attached onto the SR surfaces were disaggregated by a 0.25% trypsin/PBS solution. Next, the number of washed epithelial cells was calculated by a haemocytometer. The growth of epithelial cells was also investigated on the various SR surfaces for 96 h after seeding the CEC at lo5 cells well-‘.

In viva test

Additionally, a rabbit penetrating keratoplasty model was employed. pHEMA-grafted SR was shaped to match the topography of corneal surface during processing, 0.35mm in thickness. Next, a donor membrane was implanted by 7.5 mm trephine, which was 0.5 mm larger than the recipient bed. A suture was placed in running fashion. Following completion of the surgery, the anterior chamber was carefully reformed with heparin-containing balanced salt solution. Temporary tarsorrhaphy was occasionally performed to prevent the rabbit’s self- trauma horn occurring. Postoperative medication was not routinely administered. Clinical photomicroscopy and immunofloure stain were taken serially in time at 0, 1, 2 weeks and 10 months, postoperatively.

RESULTS AND DISCUSSION

Part I: surface properties analysis

Peroxide groups were introduced to the surface of SR by Ar-plasma treatment. Sequentially grafted polymerization was then carried out with pHEMA on the surface of SR. Results obtained from this procedure are discussed in this section.

(a) IOOOOr

loo0 / -CH3(15D) loo t II c III ‘0 I

I I I III I I I I I I I I I 0 25 50 15 100 125 150 175 200 225 250

@I 100000

1 0 25 50 75 100 125 150 175 200 225 250

I 0 25 50 75 100 125 150 175 200 225 250

m/e-(amu)

Figure 1 Negative secondary ion mass spectroscopy (SIMS) spectra of surface for silicone rubber membranes: a, control silicone rubber (SR); b, Ar-plasma treatment; c, grafted with 56pg cm-* poly(2-hydroxy ethyl methacrylate) (pHEMA).



Peroxide group determination SIMS study. Figure 1 shows the negative ion SIMS spectra for a controlled SR, Ar-plasma-treated SR, and pHEMA grafted onto SR specimens. A comparison made of the negative ion spectra of controlled SR with that of Ar-plasma-treated SR revealed that the clustering of peaks for an SR control was different from that of Ar-plasma treated SR, especially the m/e (33D) on the Ar-plasma treatment, as shown in Figure lb. After SR was treated by Ar-plasma treatment, the peak (33D) was predicted to be a peroxide group such as -0OH. Thus, the spectrum was used to confirm the introduction of a peroxide group on the SR surface. The difference between the negative ion spectrum of Ar-plasma-treated SR and that of pHEMA grafted onto SR is discussed in ‘Grafted polymerization’.

Peroxide destruction study. The surface concentration of generated peroxides is plotted in Figure 2 as a function of Ar-plasma treatment time. In this figure, the maximum value of the peroxide group was found at 60s of Ar-plasma treatment time. This result was similar to that of Ols/Cls studies. That is, this point may be the most active for plasma-induced grafted polymerization (PIP). Beyond 60 s, the value of peroxide group decreased with an increase in Ar- plasma treatment time. This occurrence could be a result of etching having occurred on the treated surfaces of SRs.

Grafted polymerization SIMS study. A decrease in the 33D peak in Figure lc was found to occur when SR was grafted with pHEMA. Whereas this increase would be caused by

800

Ar-plasma treated time (second)

Figure 2 Concentration of peroxide group on silicone rubber membrane with different treatment times for Ar- plasma by the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method.

using the peroxide group to bind a pHEMA chain with SR, 44D (-0-C=O) and 72D (-COOCC-) peaks were also found on the surface of SR grafted with pHEMA. That is, grafted pHEMA contained a carboxyl group20,21. The intensities of specific peaks for the treated SR are listed in Table 1.

ATR-FTXR study. The absorption peaks of the hydroxyl and carbonyl groups appeared at 330Ocm-l and 1720 cm-l, respectively, when pHEMA was grafted onto SR, as shown in Figure 3. Furthermore, thee intensity of the absorption peaks increased with an ’ increasing amount of pHEMA grafted onto SR.

ESCA study. The binding states and elemental compositions of SR surfaces were examined by survey scans (low resolution spectrum) and the high resolution Cls of ESCA. The survey scan (O-1000eV binding energy) was run at an analyser pass energy of 100 eV and an X-

41

r

301

(B) _.C-‘--. _, _,

0 3000 2000 lO(

Wavenumber (cm-‘)

Figure 3 The ATR-FTIR spectra for silicone rubber membrane: A, control silicone rubber (SR); B, Ar-plasma treatment; C, grafted with 56lgcm-’ pHEMA; D, grafted with 75pg cm-’ pHEMA; e, grafted with 280~gcm~’ pHEMA; F, grafted with 56Opg cmm2.

Table 1 Intensity of specific peak (amu) by secondary ion mass spectroscopy for various silicone rubber surfaces

Sample -CH3 (15D) -0OH (33D) -O-C=0 (44D) HOCC- (45D) -COOCC- (72D)

Control 300 _ - -

Ar-plasma’ _

pHEMAt 1400 3000 1700 500 2200 10 3800 3800 800

‘60 W, 200 mTorr. 60 s t 56pgcmm2.


Formation of artificial cornea from pHEMA-grafted silicone rubber membranes: S.-D. Lee et a/. 591

ray spot size of 1.6ms to determine the elemental composition of each of the SR samples. The high resolution Cls spectra were obtained at a pass energy of 25 eV and a spot size of 0.2ms. Elemental compositions, displayed as an oxygen to carbon ratio (OldCls), were obtained by making a comparison of the intensities of core-level photoelectron emission for different elements. The OlsKls ratio of SRs was altered by different Ar-plasma treatment times and the amount of pHEMA grafted onto SR was subsequently ;5tained. These results indicated that the ratio .ncreased to a maximum after 1 min Ar-plasma treatment (Figure 4), whereas these Ols/Cls ratios for SR surfaces increased with the amount of pHEMA grafted onto SRs (Table 2). This increase could have been caused by increasing the amount of the oxygen with pHEMA grafted onto SRs. Subsequently, high resolution Cls core level spectra were also examined. These spectra reflect the types of carbon functional groups on the various SR surfaces. Representative Cls spectra for three types of surfaces are shown in Figure 5. The spectra were deconvoluted into three peaks, i.e. a 285.0eV peak corresponding to carbon with no bonds to oxygen (C-H, hydrocarbons); a 286.5 eV peak corresponding to carbon with one oxygen bond (C-O, ether and alcohol); and a 289 eV peak corresponding to carbon with three bonds to oxygen (O=C-0, ester)22’23. The ratios of the fitting peaks are listed in Table 2. The hydrocarbons (C-H) of SR decreased with an increase in the amount of pHEMA grafted onto SR; however, both C-O and O=C-O peaks increased.

0.75

0.70

0.65 f I I

0.60 I

0.55 I I

0.50

0.45

0.4

2:: 0 2 4 6 8 10

Ar-plasma time (min)

Figure 4 Variation of Ols/Cls values for silicone rubber membranes with different treatment times by Ar-plasma.

Table 2 Percentage (%) of various carbon bonds by Cls high resolution electron spin microscopy for chemical analysis

Sample C-H C-o o-C=0

Control 100.00 Ar-plasma* pHEMA (55)t

77.61 15.21 6.98

pHEMA (75)t 69.98 21.24 8.78

pHEMA (28O)t 53.01 33.37 13.63

pHEMA (570)t 36.02 40.27 23.71 20.93 47.05 32.02

'60W.2OOmTorr. 60s tpgcm-2.

1

-C-H -C-H

I I

4 290 285

Eb (lev/div)

: I 290 285

Eb (lev/div)

Figure 5 High resolution spectra from electron spin microscopy for chemical analysis (ESCA) for silicone rubber membranes: A, control SR; B, Ar-plasma treatment; C, graft with 56 pg cm-’ poly(2-hydroxy ethyl methacrylate) (pHEMA); D, grafted with 75pgcm-’ pHEMA; E, grafted with 28Opg cmW2 pHEMA; F, grafted with 56Opg cme2 pHEMA.

I I I 500 1000 1500

pHEMA grafted amount (@cm-2)

Figure 6 Variation of contact angle with different amounts of grafted poly(2-hydroxy ethyl methacrylate) (pHEMA) on the silicone rubber membrane.

Contact angle study. The change in contact angles was measured by a static contact angle meter for various amounts of pHEMA-grafted SR, as shown in Figure 6. The contact angles decreased rapidly with an increase in the amount of pHEMA-grafted SRs. Furthermore, the levelling-off values (approximately 52”) were obtained for the region of 50-750pgcm-’ pHEMA on SRs. Based on these results, the wettability of SR was improved by plasma-induced grafted polymerization.

Part II: biological studies

In vitro and in vivo tests were performed in this section. CEC were attached and grown onto various surfaces of SR and the morphologies of CEC attached onto SR were subsequently observed by the in vitro tests.



Furthermore, an experiment using the eye of a rabbit was performed for the in vivo study.

In vitro test Cell attachment and growth QSSQY. CEC were isolated from the rabbit cornea. Primary CEC were seeded onto various SRs for cell attachment and growth. The attachment assay of CEC was performed on an SR control, treated by Ar-plasma SR grafted with different amounts of pHEMA and pHEMA hydrogel. The number of CEC 8 h after seeding is shown in Figure 7. This figure clearly shows that CEC could not attach itself to the surfaces of the control, Ar-plasma-treated SR and pHEMA hydrogel, whereas a large number of adherent CEC were found on SRs grafted with 5 5 pg cm-’ pHEMA. The results suggest that these surfaces provide suitable environments for CEC attachment.

The influence of CEC attached onto SR (with or without serum] was deemed negligible. On the other hand, the number of cells grown on modified SR was determined by proliferation assay for 72 h, as shown in Figure 8. This figure reveals that CEC could not grow on the control, Ar-plasma-treated SR, pHEMA hydrogel and SR grafted with 570pgcm-’ pHEMA. CEC grown onto SRs grafted with 280 pg cm-’ pHEMA showed a typical time. Beyond this point, the CEC number gradually increased until CQ 42 h and then eventually decreased. These phenomena may be caused by a few CEC attached onto SR grafted with pHEMA (570~gcm~2). On the other hand, CEC grown onto both SRs grafted with 55 pgcm-’ and 75 pgcrn-’ pHEMA showed a normal proliferation of CEC and was also confluent at 72 h. Therefore, a suitable environment for the growth of CEC onto SR was obtained from these results.

Cell morphology study. Cell morphologies were observed by inverted microscopy, as shown in Figure 9. The attachment and growth of CEC onto the control and Ar-plasma-treated SR were deemed negligible. The morphology of CEC attached onto SR grafted with 57- 75 pg cm-’ pHEMA was quite similar to that of primary CEC (Figure 9c). This similarity indicated that the modified surface provided an adequate environment for cell attachment and growth. The morphologies of

pHEMA*

pHEMA(570)

0 Serum(-)

pHEMA(280)

pHEMA(75)

pHEMA(S5)

Ar-phSlM

COIlhQl

0 2 4 6 8 IO I2

Number of cells (x 103)

The PK technique was used in this study. The implantation position of a modified membrane in the cornea is shown in Figure 10. In this case, 75 pg cm-’ of pHEMA grafted onto SR was utilized based on the in vitro test results. The results obtained from using the continued suture to tightly fix the implant and host cornea are shown in Figure 11. The implant was clearly at zero time. Next, the migration of CEC from host cornea to implant was investigated by immunofluorescence stain; in addition, the depth of the anterior chamber (AC) was measured by slit lamp microscopy. The experimental results indicated that, with time, the depth of AC gradually disappeared for controlled SR, whereas the depth of AC was maintained and the downgrowth of CEC occurred for SR grafted with pHEMA, as shown in Figure 12. This phenomenon indicated that the iris did not adhere on the surface of modified SR. Furthermore, pHEMA-grafted SRs were completely covered with CEC at postoperative week 3 (Figure 13). Subsequently, a newly formed cornea emerged when the implant was extracted from the host cornea at postoperative month 10, as shown in Figure 14. The downgrowth of CEC or stroma occurred, leading to the above results25. Moreover, the degree of transparency of a newly formed cornea increased with time.

Figure 7 The number of cells attached onto the surfaces Notably, in this study, the depth of AC could be

with various amounts of grafted poly(2-hydroxy ethyl maintained for 3 weeks and the CEC could cover SR methacrylate) (pHEMA) after 8 h. grafted with 75 pg cm-’ pHEMA at postoperative week

I I I I 0 20 40 60 80

Time of cultured (hour)

Figure 8 The proliferation of cells onto surfaces with various amounts of grafted poly(2-hydroxy ethyl methacrylate).

CEC attached onto the surfaces of control SR, Ar- plasma-treated SR and SR grafted with 570, 1250 and 1650 pgcm-’ pHEMA were poor, as evidenced by the presence of CEC pseudopodia. These morphologies are shown in Figures 9Q, b, d, e and f, respectivelyZ4. This phenomenon can be accounted for by the fact that excessive grafting of 1250 and 1650~gcm-2 pHEMA onto SR was a result of the hydrogel characteristics and subsequently inhibited the capability of CEC attachment and growth.

In vivo study

Biomaterials 1996. Vol. 17 No. 6


Figure 9 The morphologies of cells attached onto silicone grafted with 56/rg cm-* poly(Bhydroxyethyl methacrylate) 125Opg cm-* pHEMA; f, grafted with 165O/rg cm-* pHEMA.

rub (Pl

mer (SR) membrane: a, control SR; b, Ar-plasma treatment: c, IMA); d, grafted with 570pgcm-* pHEMA; e, grafted with

3. The initial stage for developing an artificial cornea was achieved on the basis of the results of this study. On the other hand, the downgrowth of CEC that occurs must be resolved for wound dressing purposes in a future study.

CONCLUSIONS

Various amounts of pHEMA grafted onto SR were obtained by varying the reaction conditions of a homobifunctional membrane. Additionally, experimental results indicated that the influence of HEMA concentration on the grafted amount is an important factor in the graft polymerization system. However, a large amount of grafted pHEMA on SR significantly

Figure 10 Implantation position of modified membrane in a rabbit cornea.



Figure 11 Slit lamp micrograph of rabbit cornea implanted with a silicone rubber membrane at zero time. Figure 13 lmmunofluorescence staining micrograph for

rabbit cornea implanted with silicone rubber membrane (75pg cm-’ pHEMA) at postoperative week 3.

Figure 12 Slit lamp micrographs of rabbit cornea implanted with silicone rubber membrane: a, control SR at postoperative week 1; b, silicone rubber membrane (75 pg cme2 pHEMA) at postoperative week 3.

affected the transparency of SR. Hence, the reaction condition was controlled to obtain good biomaterials.

ESCA spectra revealed that the Ols/Cls ratio would increase with an increase in the amount of pHEMA grafted onto SR. The peroxide group introduced on the surface of SR was confirmed by the negative ion spectra of SIMS and the DPPH method after SR treatment by Ar-plasma. The specific functional groups

Figure 14 a, Slit lamp micrograph; b, immunofluorescence staining micrograph for newly formed rabbit cornea at postoperative month 10.

and amount of peroxide groups were also determined in this work. Therefore, graft polymerization of pHEMA onto SR was carried out successfully.

The assaying of CEC attached and grown onto various SRs was also examined to study the influence of different amounts of grafted pHEMA on the capability for cell attachment and growth. The experimental results

Biomaterials 1996. Vol. 17 No. 6


indicated that 55 and 75 c(g cm-’ pHEMA grafted on SR provided a suitable environment for cell attachment and growth. Furthermore, these membranes were implanted into the rabbit cornea to confirm the biocompatibility of the membrane with the tissue. The CEC were capable of covering the implant completely in 3 weeks. In this case, however, downgrowth of CEC occurred. Therefore, heterobifunctional membranes are to be developed in a future work to prevent this downgrowth from occurring when the membrane is implanted into a rabbit cornea.

ACKNOWLEDGEMENTS 15

The authors would like to thank the National Science Council of the Republic of China for financial support of this manuscript under Contract No. NSC83-0420- 13075A-018M018, as well as VGH-NTHU Joint Research Program (VGHTH 83-021-2), Medical Research Advancement Foundation in memory of Dr Chi-Shuen Tsou, ROC.

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