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B. V. Stanzel, P. Huie, M. S. Blumenkranz, S. Binder, M. F. Marmor; Towards Prosthetic Replacement of Bruch’s Membrane: Comparison of Polyester and Electrospun Nanofiber Membranes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5085.
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Cellular replacement strategies of the RPE could be beneficial to patients with AMD, but may not be effective if Bruch's membrane (BM) has been damaged by age and/or surgery. Delivery and long term survival of these transplants may require a biocompatible substrate that patches or entirely replaces diseased BM. We have compared two candidate substrates.
Polyester transwell culture inserts (PET) are 10µm thick and permeable (purchased from Corning, Inc.). Polyamide nanofiber inserts (PN) consisted of 2 or 4µm thick electrospun nanofiber meshworks adherent to impermeable plastic (donated by Donaldson Co.). Some PN were lysed from the plastic by 100% EtOH, and then affixed onto empty frames of transwell inserts with dental wax, creating a permeable PN. Fetal human RPE cells cultured from 2 donors according to the Hu & Bok method were used in all experimental conditions, which included plastic, PET, impermeable PN, 2 and 4µm thick permeable PN. Rats were used in preliminary animal subretinal implantations.
Cells grown on impermeable substrates (PN and plastic) showed hexagonal morphology and fluid dome formation as early as 4 days postconfluence and maintained it for at least 2 weeks. Staining with ZO-1 and Claudin 3 gave a positive signal. Results differed for the permeable substrates PET and PN. On PET, cells maintained a uniformly hexagonal monolayer which gradually remelanized over a 3 months observation period. On permeable PN, the initially confluent monolayer began transforming into lattice-like clumps and most detached as an entire sheet between days 5-7. The detachment tendency was somewhat less with 4µm than 2µm thickness. Preliminary rat subretinal implantation data showed no inflammation or toxicity with PET or PN.
Permeable PET and impermeable PN support growth and differentiation of RPE cells beyond 2 weeks. However, permeable PN does not maintain RPE monolayer formation, which suggests an effect of flexibility or permeability relative to PET. It is possible that this may be resolved with other formulations of nanofiber spacing or thickness. Our preliminary data suggest gross tolerance of both PET and PN in the subretinal space. Thus PET is a promising BM substitute, and the value of PN is still indeterminate.
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