Nanoanalytical Analysis of Bisphosphonate-Induced Alterations of Microcalcifications Using a 3D Hydrogel Platform

Abstract

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased cardiovascular morbidity in patients with prior acute events, but decreased morbidity in event-free patients. We recently demonstrated that calcifying extracellular vesicles (EVs) released by cells within atherosclerotic plaques aggregate and nucleate calcific mineral. We hypothesize that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the resultant risk of plaque rupture. 3D collagen hydrogels incubated with calcifying EV mimicked fibrous cap calcification, serving as a platform to image mineral nucleation and test potential therapies for vascular calcification. This study visualized EV aggregation and formation of stress-inducing microcalcifications at single-EV resolution, via scanning electron microscopy (SEM) and atomic force microscopy (AFM). Finite element analysis (FEA) of a representative microcalcification formed in the 3D platform predicted a consequential 350% increase in fibrous cap tensile stress. Energy-dispersive x-ray spectroscopy (EDS) confirmed that EV aggregates contained calcium and phosphorous. BiP (ibandronate) treatment added on day 0 significantly decreased the size (21.5 μm2 vs. 14.2 μm2, p=0.012) and amount of EDS-detected calcium (4.32% by weight (wt%) vs. 2.36 wt%, p<0.001) and phosphorous (4.26 wt% vs. 1.94 wt%, p<0.001) comprising EV aggregates measured on day 8. However, an increase in mineral was measured when BiP was added at later time points. Mineral composition and morphology, measured with Fourier-transform infrared spectroscopy (FTIR), SEM and AFM, varied depending on the timing of BiP treatment, suggesting that BiP treatment altered microcalcification morphology and composition in a time-dependent manner. These findings support our hypothesis that BiP alter EV-driven calcification, which may help explain confounding clinical data. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.