Selective silver precipitation and malachite green uptake reveal calcium-binding sites and phospholipid involvement on calcified aortic valve thin sections

Previous study on porcine aortic valves implantated in rat subcutis showed that glutaraldehyde-Cuprolinic blue reactions (GA-CB), at low pH, induce tissue unmasking from calcium and subsequent visualization of cellsand matrix-vesicles-like bodies outlined by peculiar, CB-reactive layers; because of anionic nature and differential chemical/enzymatic extra activity, these structures were assumed to be composed by acidic phospholipids (Ortolani et al. Connect Tiss Res 2002; 43:44-55; Histochem J 34:41-50). In the present investigation, pre-embedding GA-CB reactions followed by post-embedding von Kossa silver staining (GA-CB-S) showed major metal precipitation just occurring on the pericellular CB-reactive layers, and minor one at three additional sites: (i) nuclear heterocromatin; (ii) juxtacellular, filamentous material; and (iii) collagen fibrils. Moreover, glutaraldehyde-malachite green (GA-MG) pre-embedding reactions, at lowered pH, followed by osmium post-fixation gave rise to the appearance of pericellular osmium-MG-reactive layers, which were comparable to the silver-CB-reactive ones. These data show that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface replacing cell membranes and acting as major apatite nucleator. In addition, the overall data are consistent with the concept that common steps would exist for the various pathways in normal and pathological calcification