Age-related macular degeneration is the most common form of vision loss in the elderly, and sub-retinal transplantation of autologous retinal pigment epithelium (RPE) or iris pigment epithelium to rescue diseased RPE and photoreceptors is a potential experimental therapy.1–3 Many attempts have been unsuccessful due to the lack of a proper transplantation substrate. Injection of a suspension of RPE or transplanting untethered sheets of cells may fail due to poor cell growth and orientation.4–5 Cells cultured on a substrate could address these issues. Moreover, a substrate has the added benefit of replacing diseased Bruch’s membrane and may prevent the ingrowth of new blood vessels caused by the high intraocular vascular endothelial growth factor levels associated with age-related macular degeneration.6 Many substrate materials have been attempted, including autologous lens capsule and Descemet’s membrane, resulting in either biocompatibility issues or inadequate mechanical properties to withstand a subretinal surgical procedure.7,8
Design And Methods
Bucky paper was prepared from crude preparations of single-walled carbon nanotubes synthesized by laser ablation. The crude preparation was first purified by refluxing in nitric acid (160 hours) and then centrifuged. The pellet was resuspended in potassium hydroxide solution (pH 10), and then washed twice by centrifugation/resuspension to remove and neutralize the nitric acid. The purified carbon nanotubes were then washed twice in distilled water by centrifugation/resuspension. The carbon nanotubes were formed into bucky paper by resuspending them in distilled water, then removing the water by vacuum-filtration over a cellulose filter (Figures 1A–1B). Bucky paper was prepared at a density of approximately 2,000 μg/cm2.
Figure 1. (A) Carbon nanotube bucky paper. (B) Low power scanning electron micrograph of the surface of bucky paper after fabrication. There were no visible holes in the porous mesh that would allow nutrients and waste materials to diffuse through. (C) High power scanning electron micrograph of bucky paper demonstrating the fiber-like arrangement of the carbon nanotube bucky paper.
To demonstrate that bucky paper could be used as a cell transplantation substrate, human RPE cells were cultured on its surface. Human RPE cells (ARPE-19; American Type Culture Collection, Manassas, VA) were maintained in a combination of Dulbecco modified Eagle medium (Gibco, Grant Island, NY) and Ham F-12 nutrient mixture (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in an environment containing 6.5% carbon dioxide. RPE cells were cultured onto bucky paper sterilized by ultraviolet irradiation at 13106 cells/mL. Cells were allowed to attach and grow on the bucky paper surface for 1 hour, 3 days, and 7 days prior to analysis by light microscopy and electron microscopy.
To investigate bucky paper as a substrate for retinal cell transplantation and to assess its in vivo biocompatibility, bucky paper was implanted underneath the retinas of nine rabbit eyes. Animal experiments were approved by the Stanford University Administrative Panel on Laboratory Animal Care and the care of the animals conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
New Zealand White rabbits were anesthetized with ketamine (40 mg/kg) and xylazine (5 mg/kg) administered via intramuscular injection. A standard three-port pars plana vitrectomy was performed, and a subretinal bleb was created posterior to the equator and the medullary array by injection of balanced salt solution through a 41-gauge needle (Synergetics, Inc. O’Fallon, MO). A retinotomy 1 mm in diameter was created using a pulsed electron avalanche knife9 (Carl Zeiss Meditec, Inc., Dublin, CA) and bucky paper, sterilized by immersion in 70% ethanol for 2 minutes, was inserted into the subretinal space through the retinotomy using subretinal forceps (Model 1286.R; DORC, Zuid-Holland, The Netherlands). Bucky paper was precut to fit through a 1-mm retinotomy and inserted by grasping the edge of the paper with the sub-retinal forceps, placing it through the retinotomy into the subretinal bleb and releasing it there. Because bucky paper has some inherent rigidity owing to the carbon nanotube nanostructure, it was inserted in an unrolled configuration and remained flat throughout the procedure and afterwards. After implantation, the retina was reattached by air-fluid exchange.
By light microscopy, RPE cells demonstrated normal morphology and growth patterns on the bucky paper surface, as evidenced by normal monolayer formation and overlap of cellular processes (Figure 2A). Scanning electron microscopy confirmed the presence of a confluent monolayer of cells and indicated the formation of microvilli on the apical surface (Figure 2B).
Figure 2. (A) Light micrograph of a histological cross-section of human retinal pigment epithelium cells cultured on bucky paper at day 7 (original magnification 60×). Note the single monolayer with overlapping cellular processes indicating the healthy formation of intercellular junctions. (B) Scanning electron micrograph of human retinal pigment epithelium cells cultured on bucky paper at day 7. The cells form a confluent monolayer and exhibit good growth characteristics.
Two weeks after surgical implantation, bucky paper remained flat in the subretinal space (Figure 3), with the retina remaining fully attached and exhibiting no signs of edema or inflammation. Histological analysis revealed no evidence of inflammatory cells in the region adjacent to the implanted bucky paper.
Figure 3. Bucky paper implanted in the subretinal space. Photograph taken through a surgical microscope of rabbit retina 2 weeks after bucky paper (arrow) implantation in the area of central vision. There was no appreciable necrosis, inflammation, or edema in the retina above and surrounding the bucky paper implant. Furthermore, the retina is viable and nondetached. The relationship of bucky paper to the optic nerve (asterisk) can be appreciated.
We describe the development of a novel substrate for subretinal RPE transplantation that satisfies the requirements of biocompatibility and mechanical resiliency that are needed for this type of ocular therapy. Specifically, a candidate substrate must replace a diseased basement membrane, allow for epithelial cell growth, and support the diffusion of nutrients, gasses, and waste to and from the overlying retina. Moreover, it must be rigid enough to allow for proper surgical handling and, once implanted, remain flat beneath the retina.
Carbon nanotube bucky paper, a meshwork of carbon nanotubes, was selected as a candidate substrate because of its unique characteristics.10 Being constructed entirely of carbon, bucky paper accepted the growth of cultured cells on its surface and was also accepted by the host immune system. Furthermore, carbon nanotubes have an excellent mechanical resiliency, making bucky paper resistant to folding and bending during surgical procedures and after it is implanted in the host eye. We found these qualities to be evident even in pieces as thin as the diameter of a single cell (25 μm). Finally, the thickness, density, and porosity of the carbon nanotube meshwork in bucky paper can be controlled during the fabrication process, allowing for proper gas, nutrient, and waste diffusion through its surface.
We have demonstrated that bucky paper is both a hospitable surface for cell culture and biocompatible as an implantable substrate in the rabbit eye. Unlike lens capsule and Descemet’s membrane,7–8 bucky paper did not curl and was easy to handle during intraocular surgery. Furthermore, bucky paper did not curl after subretinal implantation and a negligible inflammatory response was seen in the region adjacent to the implanted material, indicating that this bio-paper has the potential for long-term implantation. These findings pave the way for the next phase of animal experimentation, to transplant RPE or iris pigment epithelium cultured on bucky paper into the subretinal space.
Although the experiments described above indicate that no special processing of the bucky paper is required for short-term biocompatibility or cell culture, options for enhancing the biological properties of bucky paper could include the adsorption of specific extracellular matrix proteins (eg, laminin, collagen, or fibronectin), growth factors or cytokines, or growth inhibitors (eg, vascular endothelial growth factor inhibitors6 or polyvinyl alcohol). Chemical modification or functionalization of bucky paper surface could also be performed.11 These strategies may be important for a wide range of ophthalmic applications of bucky paper and tissue engineering applications that extend beyond the eye.
- Klein R. Epidemiology. In: Berger JW, Fine SL, Maguire MG, eds. Age-Related Macular Degeneration. 1st ed. St. Louis, MO: Mosby; 1999: 31–56.
- Gouras P. Transplantation of retinal pigment epithelium. In: Marmor MF, Wolfensberger TJ, eds. The Retinal Pigment Epithelium, 1st ed. New York: Oxford University; 1998:492–507.
- Thumann G, Bartz-Schmidt KU, El Bakri H, et al. Transplantation of autologous iris pigment epithelium to the subretinal space in rabbits. Transplantation. 1999;68:195–201. doi:10.1097/00007890-199907270-00006 [CrossRef]
- Durlu YK, Tamai M. Transplantation of retinal pigment epithelium using viable cryopreserved cells. Cell Transplant. 1997;6:149–162. doi:10.1016/S0963-6897(96)00142-X [CrossRef]
- Ho TC, DelPriore LV. Reattachment of cultured human retinal pigment epithelium to extracellular matrix and human Bruch’s membrane. Invest Ophthalmol Vis Sci. 1997;38:1110–1118.
- Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676. doi:10.1038/nm0603-669 [CrossRef]
- Nicolini J, Kiilgaard JF, Wiencke AK, et al. The anterior lens capsule used as support material in RPE cell-transplantation. Acta Ophthalmol Scand. 2000;78:527–531. doi:10.1034/j.1600-0420.2000.078005527.x [CrossRef]
- Thumann G, Schraermeyer U, Bartz-Schmidt KU, Heimann K. Descemet’s membrane as membranous support in RPE/IPE transplantation. Curr Eye Res. 1997;16:1236–1238. doi:10.1076/ceyr.16.12.1236.5031 [CrossRef]
- Priglinger SG, Haritoglu C, Palanker DV, et al. Pulse electron avalanche knife (PEAK-fc) for dissection of retinal tissue. Arch Ophthalmol. 2005;123:1412–1418. doi:10.1001/archopht.123.10.1412 [CrossRef]
- Dumee LF, Sears K, Schuetz J. Characterization and evaluation of carbon nanotube bucky-paper membranes for direct contact membrane distillation. J Membrane Sci. 2010;351:36–43. doi:10.1016/j.memsci.2010.01.025 [CrossRef]
- Lin T, Bajpai V, Ji T, Dai LM. Chemistry of carbon nanotubes. Aust J Chem. 2003;56:635–651. doi:10.1071/CH02254 [CrossRef]