Materials Science and Engineering C 24 (2004) 185 – 189 www.elsevier.com/locate/msec
Biocompatibility of polyimide microelectrode array for retinal stimulation Jong-Mo Seo a,b, Sung June Kim b,c, Hum Chung a,b,*, Eui Tae Kim b,c, Hyeong Gon Yu a,b, Young Suk Yu a,b a
Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Seoul National University Hospital Clinical Research Institute, 110-744 Seoul, South Korea b Nano Bioelectronics and Systems Research Center, Seoul National University, 151-742 Seoul, South Korea c School of Electrical Engineering and Computer Science, Seoul National University, 151-742 Seoul, South Korea
Abstract Artificial retina is aimed for the stimulation of remained retinal neurons in the patient with degenerated photoreceptors. Microelectrode arrays have been developed for this as a part of stimulator. To minimize the damage during ophthalmic surgery and to get better contact to retina, flexible polyimide, which can be fabricated based on semiconductor manufacturing, was selected as the substrate material. In vitro biocompatibility of polyimide microelectrode array (MEA) was tested by co-culture with human retinal pigment epithelial cells (RPE). In vivo biocompatibility was tested in the rabbit eyes. The polyimide MEA showed good affinity to human RPE and did no harmful effect. It also showed very good stability and safety in rabbit eyes by 12 weeks. D 2003 Elsevier B.V. All rights reserved. Keywords: Biocompatibility; Cell culture; Implant material; Polyimide microelectrode; Retina; Visual prosthesis
1. Introduction Photoreceptor loss due to retinal degenerative diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) is a leading cause of blindness in adult [1]. Despite a near-total loss of the macular photoreceptors, the inner nuclear and ganglion layer cells in the macula survive at fairly high rate in the patients with RP [2] and AMD [3]. Retinal prostheses have great potential in alleviating the problems and disabilities produced by these diseases [4]. The feasibility of the electrical stimulation of the remaining retinal neurons is supported by clinical studies. Controlled electrical signals applied to a small area of the retina of a blind volunteer via a microelectrode resulted in the perception of a small spot of light [5]. SiO2, Si3N4, TiN, iridium [6,7], silicone, silicon [8] and platinum electrode-embedded silicone matrices [4] were tested as substrates for retinal stimulators. Platinum elec* Corresponding author. Department of Ophthalmology, Seoul National University College of Medicine, 28 Yeongun-dong, Chongno-gu, 110-744 Seoul, South Korea. Tel.: +82-2-760-3230; fax: +82-2-741-3187. E-mail address:
[email protected] (H. Chung). 0928-4931/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2003.09.019
trode-embedded polyimide film was tried in feline eye [9], but in vitro and long-term biocompatibility was not reported. In this paper, flexible polyimide was selected as the substrate material of gold microelectrode array (MEA) to minimize the damage during ophthalmic surgery and to get better contact to retina. To evaluate the feasibility of polyimide MEA as a retinal prosthesis, in vitro and in vivo biocompatibility tests were done.
2. Experimental 2.1. Polyimide MEA Various shapes of MEAs were designed to reduce the tissue damages and to take more intimate contact to the retina. Among them, a square shape was selected for biocompatibility test. To prevent tearing of edge, MEA was designed to have rounded corners and circular holes for retinal tack. The rounded corners may also reduce the retinal tissue damage (Fig. 1). Polyimide (PI2525, HD Micro Systems) was prepared as the manufacturer’s specification and MEA was fabricated based on semiconductor
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and 20 days to identify growth pattern, morphological change and viability. On days 8 and 24, live and dead RPE were harvested and were counted in Neubauer chamber using new methylene blue staining to identify abnormal proliferation or cell death. 2.3. In vivo biocompatibility tests
Fig. 1. Design example of rectangular type polyimide MEA (NB-hole). Numbers are sizes in Am.
manufacturing technique [10]. The polyimide MEA was 18.5 Am in thickness and 3 3 mm in size. Each gold electrode was 200 200 Am in size and was spaced by 250 Am. Three different types of MEAs were used in vitro biocompatibility test: NB-3 was an early model of MEA without electrode site opening; NB-hole was a model with 200 100 Am sized holes between electrodes; and NB-o was a model with electrode site opening. 2.2. In vitro biocompatibility tests Human RPE was harvested from donated eye-bank eyes and used to evaluate the adhesion and survival on polyimide MEA. Anterior segment and retina were removed under sterile conditions by means of circumferential scleral incision 2 mm posterior to the ora serrata. Eye cup was washed with Ca2 +- and Mg2 +-free Hank’s balanced salt solution. Trypsin – EDTA was agitated within the eye by gentle pipetting to loosen the RPE cells, and then removed, centrifuged (1500 rpm, 5 min) and resuspended in 15 ml 20% fetal bovine serum (FBS)/1% penicillin – streptomycin Dulbecco’s modified Eagle’s medium (D-MEM, Invitrogen, USA, Cat. No. 12800-058). The RPE was incubated at 37 jC with 5% CO2 in flask prior to the addition of 0.25% trypsin – EDTA and incubated at 37 jC for 30 – 40 min. Each type of polyimide MEA was placed in each well of 24-well plate and 1 104 of RPE was plated on each well. The RPE cultures were incubated at 37 jC and 5% CO2 for up to 4 weeks. Microscopic examination was done after 5, 10, 15
All procedures conformed to the Association for Research in Vision and Ophthalmology (ARVO) Statement on Use of Animals in Ophthalmic and Vision Research. Lenssparing three-port pars plana vitrectomy was done in five white rabbits under general anesthesia achieved by repetitive intramuscular injection of 25 mg ketamine and 6 mg xylazine per kg of body weight. The right eye of each rabbit was used for the test and the left eye was kept intact for the control. After vitrectomy, NB-o type polyimide MEA was inserted into the eyeball through sclerotomy site. In two rabbits, polyimide MEA was stretched and fixed with conventional titanium retinal tack (Grieschaber, Schaffhausen, Swiss) on the retinal surface of visual streak. In another 3 rabbits, polyimide MEA was not fixed and kept in freely floating state in the vitreous cavity. After 1, 2, 4, 8 and 12 weeks, indirect ophthalmoscopic examination was done to evaluate the inflammatory changes or other complications in vitreous and retina. On the 8th week, electroretinography (ERG) was checked in both operated and control eyes. After 12 weeks, all rabbits were sacrificed and their eyes were enucleated to evaluate cataract and other morphological changes of eyeball. The histological change of retina was evaluated under light microscope with hematoxylin –eosin stain.
3. Results and discussion In vitro tests showed that RPE cells grew on polyimide MEA in a monolayer after 10 days and showed good affinity to polyimide MEA and exposed gold electrode (Fig. 2). After 10 days, they showed contact inhibition of proliferation, which is a usual phenomenon in primary normal cell culture. There seemed no abnormal morphological changes or no piled-up growth. Cell counts increased without dead cell on the 8th day. On day 24, however, there was 5.9 – 11.7% of dead cells in control well and that of well with polyimide MEA. There was no difference between the live cell count of control well and that of well with polyimide MEA on the 8th day and on the 24th day (chisquare test, p = 0.69). Although the fenestrated type polyimide MEA is supposed to be more biocompatible than nonfenestrated one [11], there was no difference in live cell count between the fenestrated (NB-hole) and the non-fenestrated (NB-3 or NB-o) one (chi-square test, p = 0.99). NBhole showed less dead cell count on the 24th day, just the same number of control, but this difference was not significant (chi-square test, p = 0.68).
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Fig. 2. In vitro biocompatibility tests. (a) RPE cell culture on NB-hole type polyimide MEA and control. (b) RPE cell counts on various types of polyimide MEA.
Indirect ophthalmoscopic examination of in vivo tests revealed that polyimide MEA had not induced haziness or inflammatory change of vitreous for 12 weeks after the operations (Fig. 3). Dissection of eyes also certified that there were no retinal detachment, vitreous haziness or cataract change in both groups of free-floating polyimide MEA implant and fixed polyimide MEA implant. There was no displacement of fixed polyimide MEA implant for 12 postoperative weeks. ERG showed no difference between the transplanted eye and the healthy eye. Microscopic exam revealed no histological change and no evidence of retinal
neural cell loss or inflammation (Fig. 4). In other studies concerning in vivo biocompatibility of polyimide MEA, there were mild inflammatory reactions and fibrous tissue proliferation [12,13]. However, in this study and in the other one [9], there were no inflammatory reactions and fibrous tissue proliferation. This might be due to the minimal damage to retinal tissue during insertion and relatively loose contact between the epiretinally located MEA and the retinal tissue. Polyimide is cheap, easy to produce in large quantity and has well-known biocompatibility and flexibility [14]. Polyimide has been tested as the candidate of substrate for
Fig. 3. In vivo biocompatibility tests. (a) Polyimide MEA after 12 weeks of transplantation. (b) Dissection after 12 weeks of free-floating polyimide MEA transplantation. (c) ERG of operated and control eye.
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Fig. 4. Histological comparison of retina at mid-periphery in (a) operated and (b) control eye. (c) Visual streak that was tightly approximated to polyimide MEA showed no significant microscopic damage or change, and (d) optic nerve head seemed to be normal in operated eye.
microelectrode in artificial cochlear implant [12,15], but it has not been tested extensively in retinal and associated tissues. The stability of stimulating electrode itself is also important. SiO2, Si3N4, TiN, iridium [6,7], silicone and silicon [8] showed good long-term biocompatibility in most cases except TiN. In this investigation, gold electrodeembedded polyimide film was tested for 12 weeks and also showed good result. In general, silicon substrate is too hard to modify its configuration easily. Silicone matrix is also difficult to shape into thin sheet. Polyimide is easy to handle and can be shaped in any configuration. Gold is also inert in most situations, especially in various tissues.
biomaterial for the fabrication of retinal stimulator in visual prosthesis system.
Acknowledgements This paper was supported by the Nano Bioelectronics and Systems Research Center of Seoul National University, which is an ERC supported by the Korean Science and Engineering Foundation (KOSEF).
References 4. Conclusion RPE cells grew on gold electrode-embedded polyimide film in a monolayer after 10 days of culture, and showed good affinity to it. From 10 days onward, however, they showed contact inhibition. There seemed to be no abnormal morphological changes or no piled-up growth. On days 8 and 24, there were no differences in the live cell count between the control well and the well with gold electrodeembedded polyimide MEA. There was no histological difference between control and operated eye and no evidence of retinal neural cell loss or inflammation on microscopic examination. ERG revealed no difference between the transplanted eye and the healthy eye. Gold electrodeembedded polyimide film showed good biocompatibility in vitro and in vivo test and was suitable as a candidate
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