A Novel Method for In Vitro 3D Imaging of Dissecting Pressurized Arterial Segments Using X-Ray Microtomography

A major challenge in the vascular biomechanical field is the understanding of damage and rupture properties in arteries. This understanding is limited by the complexity of arterial microstructure. Several approaches were used to investigate the mechanisms behind these complex and non-linear events. Most of these studies measured properties at the macro-scale in order to develop continuum constitutive models [29, 36, 39]. Nevertheless, the understanding of the microstructure influence seems to be the key to evidence the phenomena leading to rupture and to propose appropriate modeling approaches. In that purpose, studies combining mechanical loading and microstructural observations are crucial to investigate damage propagation in arteries.

Aorta and major proximal arteries are composed of three layers. The innermost layer, called intima, is mainly composed of endothelial cells, making contact with the blood. The media is the middle layer, composed of several musculo-elastic unit separated by elastin laminae [10]. The outermost layer is the adventitia, mainly composed of collagen. It has a significant role in the stability and ultimate strength of the artery. Aortic dissection is characterized by a sudden delamination of the aortic wall, occurring in the medial layer. It is a life threatening condition with high mortality rate [26, 32]. This event can be separated in two different mechanisms: initiation and propagation. In the majority of cases aortic dissection initiates with an intimal tear which propagates through the medial layer, hence allowing blood to flow in a false lumen. Nevertheless, intramural bleeding can also cause aortic dissection as no communication is found between these two lumina in 4% to 13% of aortic dissections [14, 19]. Usually, the intimal tear is believed to be in a radial-circumferential plane (\(\simeq \) 80 % of cases) and involves more than half the circumference of the aorta [40]. There is a lack of understanding concerning the phenomena behind the tear propagation. For more information on aortic dissection see, for instance, the review by Brunet et al. [5].

Several experimental studies investigated the behavior of arterial wall related to dissection: liquid infusion test [7, 33, 38, 41], radial tensile test [24, 36], peeling test [29, 36, 42], shear test [16, 17, 37, 48]. Although these mechanical tests allowed characterizing the rupture properties of arterial tissue, there is a lack of understanding on the early stages of aortic dissection, especially at the microstructural scale. The tension-inflation test is closer to in vivo conditions and was performed in multiple experimental studies to create an in vitro dissection [9, 13, 15, 30, 44]. Nevertheless, one of the difficulties with the investigation of dissection is that it is an intramural process which complicates observation. Combining the tension-inflation test with an appropriate imaging technique would overcome that issue and allow observing and quantifying the different steps of aortic dissection, especially in the early stages.

Different imaging techniques were used to observe arterial tissue. However, few were used in combination with mechanical tests. Recently multiphoton microscopy was often used to image arterial tissue as it allows observing collagen and elastin fibers. These studies investigated the organisation of collagen fibers structure inside the arterial wall during uniaxial and biaxial loading tests [8, 22, 47]. Although this approach provides many insights into the behavior of arterial wall at a micro-scale, the limited volume of observation (< 1 mm³) is a major limitation in the observation of aortic dissection. X-ray microtomography (micro-CT) is an alternative non-destructive imaging method with a larger field of view (1-10 mm³). This technique allows observing the 3D microstructure of soft tissue with high resolution [25, 46]. However, in situ tests on soft tissues like arteries are still scarce in the literature. In a previous study, Helfenstein-Didier et al. [18] performed in situ uniaxial tensile tests on medial layer strips of porcine thoracic aorta inside a micro-CT setup with a resolution of 4 μ m to investigate the damage initiation and propagation mechanisms. Sodium polytungstate was used as a contrast agent to observe the musculo-elastic units of the media. The results highlighted the importance of the shear delamination of the medial layers in the failure process. This conclusion was supported by histological observations on the ruptured samples. Similar observations were also presented in the study of Sherifova et al. [34] which concluded that the microstructure and especially the collagen fiber architecture but also the different types of cross-links are of first importance in the uniaxial failure of soft tissues. In another in situ study using micro-CT, Trachet et al [43] performed tension-inflation test on mice left common carotid arteries, using synchrotron-based phase-contrast imaging. High resolution images of the carotid artery were obtained with a resolution of 1.625 μ m, allowing the elastic lamellae to be segmented. The results showed that the lamellae stretch simultaneously as luminal pressure increases.

The reconstructed 3D images of the carotid artery showed clearly the media and the notch. Nevertheless, the medial lamellae could not be distinguished in all scans with the current resolution. As stated in the “Sample Preparation”, only sample 2 was quantitatively analyzed in the present study. The results of the prematurely-dissected sample 1 are displayed at the end of this section. Concerning sample 2, the scan in the unloaded configuration showed that the initial inner diameter and medial thickness of the specimen were 4.4 mm and 1.1 mm respectively. Cross-sectional views at the middle of the axially stretched sample are shown in Fig. 5 at different pressures. The evolution of the notch geometry is visible.

The notch was segmented at the different pressure steps and different information were extracted and displayed in Fig. 6. Figire 6a shows a 3D view of the segmented notch. Figure 6b shows a view in the transverse plane at 30% of the height of the notch. The crack follows the stretch of the wall; however, no propagation could be observed on the segmented images. The width and the depth of the notch were plotted in Fig. 6c and in Fig. 6d respectively. The decrease of notch height between 103 and 301 mmHg is explained by the imprecision of the segmentation method on the edges of the notch, especially at high pressures. The segmentation was also challenging for the lowest pressure as the notch was almost not visible because of the absence of pressure. The blade is curved on its edges to avoid stress concentration, explaining that the length of the notch was smaller than the blade length. This sample had an initial maximal notch depth of 182 μ m, in the very low range of usual notch depth created with our setup, likely explaining why complete dissection was not reached with our pressurization setup.

Figure 7a presents the mean axial loading at each pressure steps. Figure 7b, c and d show the inner diameter, the medial thickness, and the notch volume respectively. The latter markedly increased for low pressures and became relatively constant after, similarly to the inner diameter, and inversely to the medial thickness. It is interesting to note that the maximum variation of the lumen diameter along the vessel for all pressure steps was equal to 50 μ m.

On the graph Fig. 8, the arrow shows the presence of a micro-crack in the circumferential direction near the notch. This crack appeared on the images at 301 mmHg and continued to be visible for higher pressures.

A cross section view of the prematurely-dissected sample 1 is shown in Fig. 9. The initial inner diameter and the medial thickness of the specimen were 4.2 mm and 1.1 mm respectively. During the test, the media ruptured entirely; however, the adventitia was unruptured. The notch propagated on more than half the circumference of the artery and a considerable circumferential elastic recoil of the media was observed. The arrows highlight the presence of delamination planes at the rupture site.

This paper presented a novel experimental approach to study the mechanical events involved in the propagation of arterial dissection. In situ testing combining tension-inflation test with micro-CT allowed observing the internal structure of notched carotid arteries as an in vitro model of dissection.

In the present context, the main advantage of this imaging technique is to offer a suitable resolution/field of view compromise to enable both a global observation of the damage site (here, the notch) and lamellar-scale details such as intramural dissection planes which were evidence here around the notch. The deformation of the notch volume with the pressure followed the deformation of the wall. This is shown by the similar shape of the pressure-diameter curve (Fig. 7b) and the pressure-notch volume curve (Fig. 7d). This can also be seen by the evolution of the notch shape Fig. 6b where the notch flattened and became relatively stable after 301 mmHg. The absence of propagation of the notch is rather clear in one of our experiment. This could be explained by the lack of medial degeneration in the sample, which is usually considered as one of the main causes of aortic dissection. Nevertheless, aortic dissection can be triggered by a supra-physiological blood pressure (during weighlifting for instance [35]), meaning that maximum pressure may play a role in aortic dissection. Concerning the axial load, multiple factors could explain the decrease observed in Fig. 7a, for instance the axial pre-stretch being slightly lower than the actual in vivo stretch [2, 21]. The inner diameter measured during the experiment is consistent with the results found in the literature from tension-inflation test on healthy carotid arteries [4, 45]. In our experiment, the notch did not seem to affect the diameter as there were almost no variations in the inner diameter along the vessel; however, the depth of the notch was limited, which could explain this result.

During the inflation of the carotid artery, at the tip of the notch, a high tension stress is present in the remaining intact medial lamellae, and hence a high shear stress between intact and ruptured lamellae. The propagation of the notch in the circumferential direction, observed in Fig. 8, can be hypothesized to be a consequence of this local stress state, indicating the importance of shear delamination in dissection propagation. The same observation was made by Haslach et al. [17], where bovine aortic rings with a longitudinal notch on the intimal side were inflated until failure. The propagation of the crack occurred nearly in the tangential plane and the authors concluded that it was a consequence of the shear deformation present inside the wall during inflation. This conclusion is supported by the study of Witzenburg et al. [48] which demonstrated the very low interlamellar strength of arterial tissue, especially against shear stress. Other studies used uni-axial test to highlight the importance of shear delamination stress in the failure process [18, 34]. Using histology, these studies observed delamination planes at the rupture zone, similarly to the observation made in Fig. 9. This confirms the presence of shearing mechanisms during the propagation of the dissection in the tension-inflation test. Helfenstein-Didier et al. [18] also identified a pattern of rupture during uniaxial tension tests on medial samples. The results showed that an elementary rupture sequence repeated until complete failure: a bunch of musculo-elastic units breaks in tension (mode I), followed by a delamination (mode II) of this layer from the wall. Based on these observations, [6] showed that shear delamination strength is most likely the predominant factor in the initiation of dissection. The Fig. 9 seems to exhibit a similar failure sequence, the presence of multiple delamination planes indicates that several bunches of musculo-elastic units broke and started to delaminate from the arterial wall. At one point, the delamination shear stress between lamellae created by this mechanical state overcame the interlamellar strength and the crack propagated in the tangential plane in mode II delamination. Nevertheless, the second test did not reach failure due to low notch depth and could not confirm these observations; thus, further tests are needed.

The majority of aortic dissections involve more than half of the circumference of the aorta [40]. The same phenomenon was observed on the X-ray images of the present study (Fig. 9), confirming that the in vitro dissection obtained during the experiment is similar to those seen in clinic. Furthermore, the dissection propagated up to the boundary between media and adventitia, as described in experimental studies where the dissection is shown to propagate in the outer media [1, 28]. The fact that the adventitia was unruptured illustrates its protective role for the artery. Holzapfel et al [20] showed that the residual stress present in the arterial wall is layer-dependent and that the media, once separated from the other layers, shortens significantly. Thus, the elastic recoil of the medial layer observed in this sample is explained by the release of its tension stress initially present when the carotid artery was intact.

This work demonstrated the feasibility and relevance of the proposed experimental approach; however, the following limitations should be mentioned. First, in clinics most of the intimal tears encounter are in the transverse plane. The notch in the present study was made longitudinal as it simplified the identification of the fracture modes involved in the propagation of the dissection. In future work, we could perform this test with a transverse intimal notch to compare the influence of the notch direction on the propagation, or absence of propagation, of the defect. Another limitation is that the separation between the adventitia and the connective tissue during the preparation of the sample is complicated as there is a continuity between the two in this type of tissue. Further analysis could address the question of preferred propagation direction or fracture modes involved. Other interesting perspective might be to apply volume correlation analysis to quantify the strain of the arterial wall at different steps of the dissection; thus, these data could be used to validate complex model involving damage and rupture.

In conclusion, an experimental approach was presented to create an in vitro dissection model and observe it with micro-CT. This experimental approach allowed following the deformation of a notch and investigating the early stages of dissection propagation. The micro-CT images highlight the importance of shear delamination strengh during the propagation of the dissection. In situ testing associated with X-ray microtomography is a promising way to assess the mechanisms leading to rupture in biological tissue.