Nanostructural analysis of distinct nucleation sites in pathological mineralization

Abstract

Pathological mineralization, such as that in atherosclerosis or following transplantation of biomaterials, is associated with millions of deaths worldwide. A deeper understanding of the mechanisms underlying mineral nucleation in pathological mineralization will facilitate the development of novel bioinspired materials/techniques as therapeutics for either disease prevention or bone tissue reconstruction. The objective of this study was to perform a systematic and ultrastructural analysis of the nucleation sites of atherosclerotic mineralization from the viewpoint of material science. Histological staining, ultrastructural analysis, and selected area elemental mapping of human atherosclerotic tissues revealed two different nucleation sites in a collagenous and a lipidic area, respectively, comprising phospholipidic large vesicles (average 827 ± 470 nm) and non-vesicular phospholipids. In both sites, the initial minerals were identified to be amorphous calcium phosphate (ACP), which then transformed into hydroxyapatite (HAp). Mineralization assays in vitro was performed by incubating non-vesicular low- (LDL) and high- (HDL) density lipoprotein and cholesterol in 2 mM CaCl₂ for 2 days, without additional phosphate. Calcium ions in the formed minerals, measured using atomic absorption (AA) spectroscopy, showed that only LDL was mineralized. We then evaluated the mineralization of the major phospholipids, phosphatidylserine (PS) and phosphatidylcholine (PC) under the same conditions. The results of AA and mineral dry weight showed that PS was more abundantly mineralized than PC, most likely due to its higher hydrolysis rate. Similar to the in vivo findings, PS mineralization initially formed ACP, which then crystallized into HAp. Taken together, these results indicated that atherosclerosis involves two distinct nucleation sites and that non-vesicular phospholipids were one of the major nucleation sites for atherosclerotic mineral formation.