Reengineering of aged Bruch's membrane to enhance retinal pigment epithelium repopulation

PURPOSE. An earlier study showed that age-related changes in the inner collagen layer (ICL) inhibit RPE cell repopulation of human Bruch's membrane. The present study was undertaken to determine the effect of cleaning and/or an extracellular matrix (ECM) protein coating on the reattachment, apoptosis, proliferation, and final surface coverage of the transplanted RPE cells. METHODS. Explants of aged Bruch's membrane with ICL exposed were prepared from five human cadaveric eyes ( donor ages, 69 - 84 years) and treated with Triton X-100 and/or coated with a mixture of laminin ( 330 mug/mL), fibronectin ( 250 mug/mL), and vitronectin ( 33 mug/mL). Viable human fetal and ARPE-19 cells ( n = 15,000) were plated onto the surface and the RPE reattachment, apoptosis, and proliferation ratios were determined on the modified surfaces. Cells were cultured up to 17 days to determine the surface coverage. Ultrastructure of the modified Bruch's membrane and RPE morphology were studied with transmission and scanning electron microscopy. RESULTS. Reattachment ratios of fetal human RPE and ARPE-19 cells were similar on aged ICL (41.5% +/- 1.7% and 42.9% +/- 2.7%, P > 0.05). The reattachment ratio increased with ECM protein coating and decreased with detergent treatment. Combined cleaning and coating restored the reattachment ratio of the fetal RPE cells, but failed to increase the reattachment ratio of ARPE-19 cells. The highest apoptosis was observed on untreated ICL. Cleaning and the combined procedure of cleaning and ECM protein coating decreased fetal RPE cell apoptosis. Only RPE cells plated on cleaned or cleaned and ECM-coated ICL demonstrated proliferation that led to substantial surface coverage at day 17. CONCLUSIONS. Age-related changes that impair RPE repopulation of Bruch's membrane can be significantly reversed by combined cleaning and ECM protein coating of the ICL. Development of biologically tolerant techniques for modifying the ICL in vivo may enhance reattachment of the RPE and its repopulation of aged ICL.