Regeration of the Inner Collagenous Layer (ICL) of Human Bruch's Membrane by Cleaning and Extracellular Matrix Protein Coating: Morphometric Evidence

Abstract

Abstract: : Purpose: We have previously shown that increased cross-linking of thickened collagen fibers and accumulation of large extracellular debris characterize aging of the ICL of human Bruch's membrane. Here we demonstrate that cleaning and/or extracellular protein recoating of the native collagen framework of aged (>60 years old) human Bruch's membrane restores the architecture of this layer. Methods: Explants of peripheral ICL were prepared from 4 aged donor human cadaver eyes (63-89 years old). Triplicate explants were cleaned by treatment with 0.1% Triton-X / 0.1% sodium citrate, and/or resurfaced with an extracellular matrix protein mixture (ECM-P) containing laminin (330µ/ml), fibronectin (250µ/ml), and vitronectin (33µ/ml). Random SEM images were analyzed morphometrically using NIH image analysis software to determine the following morphometrical parameters in each group: total pore area (nm² per µ²), number of pores (per µ²), average diameter of collagen fibers (nm), fractal dimension of collagen fibers, area of deposits (nm² per µ²), number of deposits (per µ²) and percent deposit coverage of the surface (%). Morphometrical data compared with untreated samples from the same donors. Results: The number of pores within the ICL increases slightly (5.9%) by cleaning but decreases by cleaning and extracellular protein coating (21.7%). Detergent treatment increases the diameter of individual collagen fibers (26.9%) and results in more individual fibers (11.3 % increase) by breaking collagen cross-links. 41.9% of the deposits are removed with cleaning. Cleaning followed by extracellular protein coating doubles the amount of matrix proteins on the collagen framework without increasing the occupied area (66.9%). Conclusions: Cleaning and extracellular protein coating of the Bruch's membrane can reestablish its native structure. Regeneration of Bruch's membrane may constitute an important step in reconstructive approaches for AMD and other aged-matrix diseases.

Keywords: Bruch's membrane • age-related macular degeneration • extracellular matrix