Detection of hydroxyapatite in calcified cardiovascular tissues

Abstract

Objective The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. Methods A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. Results Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. Conclusion Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases.

Three-dimensional cell culturing by magnetic levitation

“…Indeed, the MLM has been successfully used to make 3D cultures with all cell types tested to date, including cell lines, stem cells and primary cells 13,[17][18][19][20][21][22] (Table 1). The most basic application of the MLM is to culture 3D cell cultures under different biochemical or environmental conditions, and then analyze them using common biological research techniques, such as IHC 17,18 and western blotting 19 .…”

“…Once the structures are fully formed and become competent (Step 10), compounds can be added to each well at specific concentrations. At a specific time point, the 3D cultures can then be assayed similarly to 2D cultures with tests such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 17 , IHC 17,[18][19][20][21][22][23] or western blotting 19 . Other, more complex experiments including cocultures 17,18 can be conducted as well with the MLM.…”

Dietary L-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

“…37 Because of its hexagonal crystallographic structure, hydroxyapatite has two crystal planes: a-planes and c-planes. 18,38,39 The a-planes are rich in calcium ions and are positively charged, whereas the c-planes are rich in phosphate ions and are negatively charged.…”

Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation

“…These cells interact, self-assemble, and proliferate without the need or influence of an artificial extracellular matrix; for example, magnetically levitated human glioblastoma cells showed greater proliferation and protein expression when they were magnetically levitated than when they were plated in 2D. 49,50 This technique has also been used to assemble models of cardiac SMCs, 51 as well as cocultures of endothelial cells with adipose stem cells with in vivo-like vascular endothelial and adipose architecture. 52 These attributes make magnetic levitation an attractive method for creating 3D multitype cocultures.…”