title: A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.
abstract: Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell®). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australia.|Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia.|Queensland Eye Institute, South Brisbane, Queensland, Australia.
authors.forename: Audra M A
authors.initials: AMA
authors.lastname: Shadforth
authors.affiliation: Queensland Eye Institute, South Brisbane, Queensland, Australia.
authors.forename: Shuko
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia.|Queensland Eye Institute, South Brisbane, Queensland, Australia.
authors.forename: Christina
authors.initials: C
authors.lastname: Theodoropoulos
authors.affiliation: School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australia.|Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia.|Queensland Eye Institute, South Brisbane, Queensland, Australia.
authors.forename: Neil A
authors.initials: NA
authors.lastname: Richardson
authors.affiliation: Queensland Eye Institute, South Brisbane, Queensland, Australia.|Faculty of Health Sciences, University of Queensland, Herston, Queensland, Australia.|Faculty of Science and Engineering, Queensland University of Technology, Brisbane, Queensland, Australia.|Australian Institute of Bioengineering and Nanotechnology, University of Queensland, St Lucia, Queensland, Australia.|Faculty of Science, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Traian V
authors.initials: TV
authors.lastname: Chirila
authors.affiliation: School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australia.|Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia.|Queensland Eye Institute, South Brisbane, Queensland, Australia.
authors.forename: Damien G
authors.initials: DG
authors.lastname: Harkin
mesh_terms: D000943:Antigens, Differentiation / Q000096:biosynthesis*; D016570:Bruch Membrane; D002460:Cell Line; D005136:Eye Proteins / Q000096:biosynthesis*; D005349:Fibroins; D005786:Gene Expression Regulation; D006801:Humans; D008268:Macular Degeneration / Q000378:metabolism / Q000628:therapy; D008422:Materials Testing; D008567:Membranes, Artificial; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*
chemical_list: D000943:Antigens, Differentiation; D005136:Eye Proteins; D008567:Membranes, Artificial; D005349:Fibroins
keywords: Bombyx mori; Bruch's membrane; biomaterial; cell culture; retinal pigment epithelium; silk fibroin