Organ Culture Model of Aortic Valve Calcification

Abstract

A significant amount of knowledge has been gained with the use of cell-based assays to elucidate the mechanisms that mediate heart valve calcification. However, cells used in these studies lack their association with the extra-cellular matrix or the influence of other cellular components of valve leaflets. We have developed a model of calcification using intact porcine valve leaflets, that relies upon a biological stimulus to drive the formation of calcified nodules within the valve leaflets. Alizarin Red positive regions were formed in response to lipopolysaccharide and inorganic phosphate, which could be quantified when viewed under polarized light. Point analysis and elemental mapping analysis of electron microscope images confirmed the presence of nodules containing calcium and phosphorus. Immunohistochemical staining showed that the development of these calcified regions corresponded with the expression of RUNX2, osteocalcin, NF-kB and the apoptosis marker caspase 3. The formation of calcified nodules and the expression of bone markers were both inhibited by adenosine in a concentration-dependent manner, illustrating that the model is amenable to pharmacological manipulation. This organ culture model offers an increased level of tissue complexity in which to study the mechanisms that are involved in heart valve calcification.

Challenges of aortic valve tissue culture – maintenance of viability and extracellular matrix in the pulsatile dynamic microphysiological system

“…Due to the complex and extreme microenvironment of heart valves, it remains challenging to introduce a physiological in vitro model system reflecting these conditions. Application of 2D-cell cultures allow basic experimental setups [ 9 , 21 – 23 , 26 ] but there are non-representative model attributes such as physiological cell–cell and cell–matrix interaction as well as biomechanical and -chemical microenvironmental influences [ 6 , 25 , 41 , 43 , 58 , 78 ]. Limitation of calcification in contrary is possible by adding pro-calcifying or osteogenic media [ 79 – 81 ].…”

“…The presented study describes a novel MPS for intermediate sized AV tissue incubation of both porcine and human origin under pulsatile dynamic conditions coupling the pneumatic pump system to tissue incubation chamber. Static incubation inheres model disadvantages because biomechanical simulation of extreme AV microenvironment is not reflected [ 39 , 41 , 58 ]. AV tissue segments of intermediate size were applied that allow yielding of four to five samples per leaflet in parallel.…”

“…The positive stained area was calculated with Fiji . Alizarin red staining allows investigation of AV tissue calcification [ 15 , 19 , 35 , 41 , 58 , 74 ]. All histological stainings were performed according to standard protocols.…”

Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles

“…However, enrolment may lead to an insufficient medium supply, leading to a decrease in degeneration and a loss of layer-dependent progression of calcification [39]. Enrolment could be inhibited by pinning the valves to the base of the tissue culture plate [39,40]. But even pinning did not always result in the specific calcification pattern within the valve seen in CAVD, i.e., the calcification was not primarily associated with the fibrosa of the valve leaflets [40].…”

Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview

“…Chester et al (2021) developed another model with whole leaflets where calcification is not induced by osteogenic media, but uses the combination of lipopolysaccharide and inorganic phosphate, to initiate and drive the calcification process by an inflammatory response. One of the advantages is the extensive histological investigation of the calcifying leaflets–both qualitative and quantitative ( Chester et al, 2021 ).…”