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T.H. Tezel, K. Sonmez, L.V. Del Priore, H.J. Kaplan; Tissue Reconstruction in AMD: Breaking the Collagen Crosslinks Increases RPE Reattachment Rate Onto Aged Inner Bruch's Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1212.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Successful cellular transplantation in age–related macular degeneration (AMD) requires modification or replacement of damaged Bruch’s membrane (BM), and identification and use of the optimal cell source. We have shown previously that BM can be cleaned and resurfaced to increase RPE attachment and proliferation. Herein we determine the effects of breaking collagen crosslinks and enzymatic lipolysis on the fate of ARPE–19 cells seeded onto aged inner Bruch’s membrane. Methods: Explants of human peripheral inner collegen layer (ICL) were prepared from 4 aged (>60) cadaver eyes. Triplicate sets were treated as follows: (1) removal of lipids by hydrolysis with lipoprotein lipase (LPL, 70U/ml), (2) collagen crosslink breaking with N–phenyl–thiazolium bromide (PTB, 0.1M), (3) resurfacing with an extracellular matrix protein (ECM–P) mixture containing laminin (330µ/ml), fibronectin (250µ/ml), and vitronectin (33µ/ml), and (4) a combination of all three treatments. Synchronized ARPE–19 cells (15K viable cells/well) were plated onto each surface. The effect of treatments on photoreceptor, RPE and choroidal cell viability, the RPE reattachment and apoptosis rates were determined. Results: On untreated ICL, the plating efficiency for ARPE–19 was 34.6±3.1%. Plating efficiency increased with ECM–P coating of ICL (52.2±3.1%, p<0.01) but did not increase significantly with LPL treatment (44.2±2.6%, p<0.05). PTB treatment alone (69.2±6.7%) or in combination with LPL/ECM–P (85.4±8.8%) increased reattachment rate significantly (p<0.01). The apoptosis rate for ARPE–19 cells on aged ICL (386/100,000) was reduced by ECM–P coating (20/100,000). Similarly, enzymatic lipolysis with LPL (23/100,000), PTP treatment alone (24/100,000), and PTB in combination with LPL and ECM–P treatment (20/100,000) significantly reduced RPE apoptosis rate (p<0.01). LPL and PBT treatments did not significantly alter the photoreceptor, RPE and choroidal cell viability for up to 240 minutes (p>0.05). Conclusions: Nonenzymatic cross–linking, which is known to occur in human BM as a function of advancing patient age, may be partially responsible for the age–related decline in RPE attachment and resurfacing of aging human BM. Breaking the crosslinks increases the reattachment and survival of ARPE–19 cells on aged human ICL. This treatment strategy may facilitate resurfacing of aged human Bruch’s membrane after submacular surgery in AMD.
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