The rupture of an atherosclerotic plaque cap overlying a lipid pool and/or necrotic core can lead to thrombotic cardiovascular events. In essence, the rupture of the plaque cap is a mechanical event, which occurs when the local stress exceeds the local tissue strength. However, due to inter- and intra-cap heterogeneity, the resulting ultimate cap strength varies, causing proper assessment of the plaque at risk of rupture to be lacking. Important players involved in tissue strength include the load-bearing collagenous matrix, macrophages, as major promoters of extracellular matrix degradation, and microcalcifications, deposits that can exacerbate local stress, increasing tissue propensity for rupture. This review summarizes the role of these components individually in tissue mechanics, along with the interplay between them. We argue that to be able to improve risk assessment, a better understanding of the effect of these individual components, as well as their reciprocal relationships on cap mechanics, is required. Finally, we discuss potential future steps, including a holistic multidisciplinary approach, multifactorial 3D in vitro model systems, and advancements in imaging techniques. The obtained knowledge will ultimately serve as input to help diagnose, prevent, and treat atherosclerotic cap rupture.

Rupture of the cap of an atherosclerotic plaque can lead to thrombotic cardiovascular events. It has been suggested, through computational models, that the presence of microcalcifications in the atherosclerotic cap can increase the risk of cap rupture. However, the experimental confirmation of this hypothesis is still lacking. In this study, we have developed a novel tissue-engineered model to mimic the atherosclerotic fibrous cap with microcalcifications and assess the impact of microcalcifications on cap mechanics. First, human carotid plaque caps were analyzed to determine the distribution, size, and density of microcalcifications in real cap tissue. Hydroxyapatite particles with features similar to real cap microcalcifications were used as microcalcification mimics. Injected clusters of hydroxyapatite particles were embedded in a fibrin gel seeded with human myofibroblasts which deposited a native-like collagenous matrix around the particles, during the 21-day culture period. Second harmonic multiphoton microscopy imaging revealed higher local collagen fiber dispersion in regions of hydroxyapatite clusters. Tissue-engineered caps with hydroxyapatite particles demonstrated lower stiffness and ultimate tensile stress than the control group samples under uniaxial tensile loading, suggesting increased rupture risk in atherosclerotic plaques with microcalcifications. This model supports previous computational findings regarding a detrimental role for microcalcifications in cap rupture risk and can further be deployed to elucidate tissue mechanics in pathologies with calcifying soft tissues.

A significant amount of vascular thrombotic events are associated with rupture of the fibrous cap that overlie atherosclerotic plaques. Cap rupture is however difficult to predict due to the heterogenous composition of the plaque, unknown material properties, and the stochastic nature of the event. Here, we aim to create tissue engineered human fibrous cap models with a variable but controllable collagen composition, suitable for mechanical testing, to scrutinize the reciprocal relationships between composition and mechanical properties. Myofibroblasts were cultured in 1 × 1.5 cm-sized fibrin-based constrained gels for 21 days according to established (dynamic) culture protocols (i.e. static, intermittent or continuous loading) to vary collagen composition (e.g. amount, type and organization). At day 7, a soft 2 mm ∅ fibrin inclusion was introduced in the centre of each tissue to mimic the soft lipid core, simulating the heterogeneity of a plaque. Results demonstrate reproducible collagenous tissues, that mimic the bulk mechanical properties of human caps and vary in collagen composition due to the presence of a successfully integrated soft inclusion and the culture protocol applied. The models can be deployed to assess tissue mechanics, evolution and failure of fibrous caps or complex heterogeneous tissues in general.

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture mechanics of the heterogeneous, highly collagenous plaque tissue, and how this is related to the tissue's fibrous structure, are not known yet. Existing pipelines to study plaque mechanics are limited to obtaining only gross mechanical characteristics of the plaque tissue, based on the assumption of structural homogeneity of the tissue. However, fibrous plaque tissue is structurally heterogeneous, arguably mainly due to local variation in the collagen fiber architecture. The mechano-imaging pipeline described here has been developed to study the heterogeneous structural and mechanical plaque properties. In this pipeline, the tissue's local collagen architecture is characterized using multiphoton microscopy (MPM) with second-harmonic generation (SHG), and the tissue's failure behavior is characterized under uniaxial tensile testing conditions using digital image correlation (DIC) analysis. This experimental pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue. The obtained knowledge is key to better understand, predict, and prevent atherosclerotic plaque rupture events.