Understanding the biological and physical effects of sclerotherapy

Abstract

Sclerotherapy is currently employed to effectively treat varicose veins, and relies on the injection of liquid or foamed agents causing endothelial wall damage, vessel shrinkage, and subsequent neovascularization. Pre-clinical in vitro studies are conducted to characterize the performance of sclerosing agents; however, they often do not replicate physiologically relevant physical and biological conditions. In this study, three models have been developed in order to gain a more comprehensive understanding of the physical and biological effects of sclerosing agents. These include: (i) 2D in vitro models, (ii) ex vivo models, and (iii) 3D vein-on-a-chip (VOC).

The 2D in vitro model has been employed to quantify the efficacy of sclerosing agents onto a monolayer of endothelial cells. The model allowed investigating the effect of clinically-relevant parameters, including different administration conditions. Moreover, the first systematic comparison of the biological performance of different polidocanol-based sclerosing foam formulations (PEM and physician compounded foams) was carried out, including an attempt to correlate biological effects with foam physical properties. It has been demonstrated that PEM was the most effective foam at disrupting the endothelial layer in a variety of tests and over different timescales of treatment. This was attributed to the slower drainage dynamics of PEM compared to physician compounded foams, and – potentially – to the enhanced polidocanol mobility conferred by its gas formulation.

Subsequently, a quantitative microscopy technique was employed to quantify the level of disruption of cell membranes subject to sclerotherapy. With this method, it was found that exposure to sclerosants causes changes to the lipid packing of cell membranes. Therefore, measurement of the variations in membrane lipid packing was employed to evaluate the biophysical effects of sclerosing agents. Findings were generally in agreement with the results obtained from in vitro efficacy tests. The ability of POL to penetrate and perturb the membrane lipid bilayer was found to be concentration dependent. Similar results were obtained comparing foamed and liquid POL.

Whilst 2D models enabled the investigation of the effect of sclerosants at the cellular level, they did not reproduce the 3D architecture and fluid dynamic environment typical of a varicose vein. Therefore, they were not suitable for investigating the interaction of sclerosing agents with blood in a biomimetic environment. For this reason, ex vivo models have been developed using umbilical cord veins, in order to quantifying sclerosant-induced disruption of the vessel wall using a colorimetric assay. Experiments were carried out using both liquid and foamed sclerosants, in either static or dynamic conditions, and in the presence of blood. When blood was perfused through the umbilical vein, all treatment methods presented similar efficacy.

Afterwards, 3D Vein-on-a-chip (VOCs) models lined with human endothelial cells were developed. Models replicated the architecture of physiological and varicose veins. VOCs were produced in polydimethylsiloxane (PDMS) applying 3D-printing and soft-lithography techniques, and were employed for evaluating the mechanical properties of foams. A protocol was designed to replicate the clinical administration process, using physiologically relevant vein diameter, geometry, and inclination angle (i.e. replicating leg elevation), and reproducing bulk physical properties of blood. The vein-on-a-chip models and experimental methods developed in this study provide a novel technology platform to measure the behaviour of different formulations of sclerosing foams, at physical conditions that resemble their clinical administration.

Despite PDMS-based VOCs provide a useful model to investigate the flow behavior of sclerosing foams and its relation to biological performance, they do not fully replicate the physical properties of both vascular and extra-vascular compartments of a vein. In order to overcome this limitation, the development of hydrogel-based models has been investigated. A method has been developed to manufacture channel geometries inside a hydrogel-based scaffold.

Overall, these technological developments and research findings can form the basis for a novel technological pipeline to accelerate clinical translation and innovation of sclerosing agents.

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