# In-situ compression test on the human intraluminal thrombus

Joris Léonet, Jérôme Vicente, Valérie Deplano

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Joris Léonet, Jérôme Vicente, Valérie Deplano. In-situ compression test on the human intraluminal thrombus. Multidisciplinary Biomechanics Journal, 2025, 50th congress of the.., 10.46298/mbj.16177. hal-05191729v2

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Submitted on 23 Oct 2025

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# In-situ compression test on the human intraluminal thrombus

Joris Léonetª, Jérôme Vicenteᵇ, Valérie Deplanoª*

a Aix-Marseille Univ, CNRS, Centrale Méditerranée, IRPHE UMR 7342, Marseille, France

b Aix-Marseille Univ, CNRS, IUSTI UMR 7343, Marseille, France

* Corresponding author: valerie.deplano@uni-amu.fr

Received date: 04/04/2025

Accepted date: 27/06/2025

Publication date: 27/10/2025

**Keywords:** Thrombus, histology, stress-relaxation, synchrotron

© 2025 The Authors
Licence CC-BY 4.0
Published by Société de Biomécanique

## 1. Introduction

The intraluminal thrombus (ILT) is a porous structure composed of a fibrin matrix including blood cells, and plasma proteins. It is present in most abdominal aortic aneurysms (AAAs). The ILT consists of three layers, whose composition and structure vary with aging, leading to permeability gradients. Its quantitative morphological and poro-mechanical features at microscale are scarce (Léonet et al., 2025). Several studies hypothesize that its role could lead to AAA rupture due to wall hypoxia (Vorp et al., 2001). However, this concept is still under debate. Characterize the microstructure and the mechanics of ILT is thus of major interest to bring new lighting on AAA rupture. Using histology, we qualitatively characterized the ILT structure and composition (Léonet et al., 2022). Moreover, compression tests were conducted on ILT sample showing a visco-hyperelastic behavior but without making a link with the microstructure (Léonet et al., 2023). To go further, the objectives of the present study are therefore (i) to quantify the amount of cells and tissue on several human ILT, (ii) to link morphological and mechanical properties with in-situ compression test under synchrotron X-ray micro-CT.

## 2. Methods

### 2.1 Specimens

ILT were extracted from 18 AAAs of human donor and preserved in Dulbecco's Modified Eagle Medium

(DMEM). In total, 17 ILT were used to quantify the composition using histology. Two squared samples from a single ILT composed by 2 pieces (T1, T2)—one with luminal only (S1) the other with luminal and medial layers (S2),—were used for in-situ compression testing via synchrotron.

### 2.2 Histology and immunohistochemistry

After extraction, a portion of each ILT was excised for histological and immunohistochemical analyses conducted at La Timone Hospital (AP-HM, Marseille, France). ILT were first immersed in 10% buffered formalin bath for tissue fixation, and paraffin-embedded. Consecutive sections were cut to thickness at 4µm in order to visualize the three ILT layers and stained with Hematoxylin and Eosin (HE), and anti-CD-61. The amount of red blood cells (RBC), white blood cells (WBC), fibrin, and porosity was assessed using HE staining, while platelets (PLT) using CD-61 staining. The cellular quantification was performed using QuPath software.

### 2.3 Synchrotron X-ray micro-CT

Squared samples with a section of 6 x 6 mm and 5 mm in thickness were cut preserving all three layers. Then, samples were immersed in chamber filled with DMEM and placed within an homemade compression setup. Samples were imaged using phase-contrast synchrotron radiation at ANATOMIX beamline (SOLEIL,
Saint-Aubin, France). A total of 2000 projections were taken at an effective pixel size of 3.09 µm, at 30 keV, with an exposure time of 100 ms. In total, 3 scans were taken for each sample without compression, at 15%, and 45% of deformation using unconfined stress-relaxation test. A pre-stress of 0.02N was used followed by 5 preconditioning cycles of 15% deformation at 0.02 mm/s. Then, a ramp of 15% was applied at 0.02 mm/s followed by a 60 min relaxation time. Finally, a 45% compression ramp was applied with the same parameters.

# 3. Results

## 3.1 Cellular quantification

The fractions of PLT, fibrin, RBC, WBC, and pores in ILT layers were analyzed. Fibrin was the main component, ranging from 74% in the luminal to 96% in the abluminal layer. The medial layer showed the highest porosity, 15%, compared to less than 4% in other layers. Most cells were found in the luminal layer: RBC (20%), WBC (2%), and PLT (35%). In the medial and abluminal layers, RBC and WBC were <1% and <0.15%, while PLT remained notable at 9% and 5% respectively.

## 3.2 In-situ stress-relaxation

Figure 1 presents the results of the *in-situ* unconfined stress-relaxation experiments. Regarding S2 the morphology of each ILT layer is shown without compression, revealing a luminal layer composed of loose fibrin fibers in contrast to the medial layer, which appears denser with larger pores. Under 15% compression, the first fibrin fibers in the luminal layer begin to compress. At 45% deformation, both the luminal and medial layers are compressed, with the fibrin fibers in the luminal layer becoming fully compacted and some medial layer pores closing. These results can be linked to the mechanical properties. When compressing at 15%, only the luminal, the softer layer, is involved explaining the low forces recorded. When applying 45% of compression, the two layers are compressed justifying the high forces compared to the first ramp. We observed some differences between the curves even if the sample came from the same ILT, explaining by the ILT heterogeneous structure and the difference between S1 and S2. Finally, we identified a non-linear viscoelastic behavior.

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**Figure 1.** In-situ experiment. (A.) Sample preparation on 2 ILT pieces (T1 and T2) coming from the same patient; (B.) ILT sample 2 microstructure without deformation; (C.) at 15% deformation; (D.) at 45% deformation; (E.) stress-relaxation curves of the 2 samples. L and M stand for luminal and medial, respectively.

# 4. Discussion

Significant differences were found between layers in porosity—higher in the medial layer—and fibrin levels, higher in the abluminal layer. Cell quantification showed most cells in the luminal layer, suggesting its permeability as observed previously (Léonet et al., 2022) and suggesting ILT may contribute to aortic wall hypoxia. A link between morphology and mechanical behavior was established using synchrotron-based *in-situ* compression. However, there is still a need of a theoretical model to extract viscoelastic properties.

# 5. Conclusion

For the first time, unconfined compression tests under synchrotron radiation were performed on human ILT samples. This ongoing study aims to expand the ILT sample set, conduct quantitative morphological analyses on synchrotron images, and develop a robust ILT model.
## Acknowledgements

The authors thank Mariangela de Masi-Jacquier for providing the samples, Guillaume Daniel and Jonathan Perrin from SOLEIL synchrotron, who installed the experimental setup and assisted in configuring the acquisition parameters respectively, as well as Guillaume Bravais and Sofiane Terzi from NOVITOM, who handled the data acquisition and image reconstruction.

## Conflict of Interest Statement

None.

## Funding

Initiative d'Excellence d'Aix-Marseille Université-AMIDEX (AMX-19-IET-010).

## Contributor Roles

JL: Conceptualization, Methodology, Formal analysis, Writing original draft; JV: Supervision; VD: Funding acquisition, Supervision, Validation, Writing-review & editing.

## References

* Léonet, J., Vicente, J., et al. (2022). Morphological and poro-mechanical characterization of thrombi from aortic aneurysm. In 47ᵉ congrès de la Société de Biomécanique, 5186-5188. Retrieved from https://hal.science/hal-03859503

* Léonet, J., Vicente, J., et al. (2023). Contrast-enhanced micro-CT protocol for in-situ mechanical experiments of aortic thrombi. In 48ᵉ congrès de la Société de Biomécanique, S125-S127. Retrieved from https://hal.science/hal-05191729v1

* Léonet, J., Vicente, J., et al. (2025). From characterization to model of thrombi: A literature review. *Heliyon* [Preprint]. https://doi.org/10.2139/ssrn.4969435

* Vorp, D. A., Lee, P. C., et al. (2001). Association of intraluminal thrombus in abdominal aortic aneurysm with local hypoxia and wall weakening. *J. Vasc. Surg., 34*(2), 291-299. https://doi.org/10.1067/mva.2001.114813