Previous studies have demonstrated that several structural and molecular alterations occur within human Bruch’s membrane as a function of age. These changes, which disrupt the delicate molecular architecture of Bruch’s membrane, include (1) structural changes in the main collagen framework, including cross-linking and deposition of long-spaced collagen
38 ; (2) qualitative and quantitative changes in the native ECM molecules
39 ; (3) deposition of abnormal extrinsic molecules,
40 ; and (4) macromolecular changes in the structure of Bruch’s membrane, such as drusen formation, calcifications, and cracks or loss of inner layers due to inadequate basal membrane regeneration, as in geographic atrophy.
31 15 Additional structural alterations can be induced by submacular surgery, because excised neovascular membranes in AMD eyes contain fragments of the basal lamina and deeper layers of Bruch’s membrane, thus exposing the ICL and perhaps other layers. The chemical treatments we used to reengineer the aged human Bruch’s membrane act by (1) liquefying and extracting membranous lipoprotein debris from the ICL to expose ECM protein receptors on native collagen fibers
40 41 ; (2) reestablishing the native collagen framework by dissolving long-spacing collagen
42 and breaking collagen cross-links
43 ; and (3) polymerizing a layer of ECM proteins onto the rejuvenated core collagen matrix of Bruch’s membrane.
44 A nonionic detergent (Triton X-100) was used to extract membranous debris from the aged Bruch’s membrane while preserving the anionic glycosaminoglycan bridges between the collagen fibrils and the native structure of collagen.
45 At the concentrations we used, Triton X-100 dissolved the membranous debris of age-related photoreceptor outer segments
46 without disrupting the ultrastructure of the matrix.
47 It also did not interfere with the subsequent adhesion of ECM proteins to the collagen fibers
48 and allowed them to polymerize in their native form on the collagen matrix.
49 Detergent treatment before ECM protein coating avoided the binding of ECM molecules to lipoprotein debris with a consequent abnormal configuration.
50 The reducing agent sodium citrate was added to solubilize the lipid debris and to facilitate the breakdown of age-related pentosidine cross-links between collagen fibers.
51 In theory, the removal of the lipoprotein debris and the secondary increase in anionic binding sites may induce a shift toward hydrophilicity and increased hydraulic conductivity of the ICL.
52 Taking all results together, we believe that the chemical treatments we used to reengineer the aged human Bruch’s membrane acted by liquefying and extracting membranous lipoprotein debris from the ICL to expose ECM protein receptors on native collagen fibers,
40 41 thus reestablishing the native collagen framework by dissolving long-spacing collagen
42 and breaking collagen cross-links.
43 This allowed proteins subsequently placed on this surface to polymerize onto the rejuvenated core collagen matrix of Bruch’s membrane.
44