Atherosclerotic plaque rupture in carotid arteries is a major cause of cerebrovascular events. Plaque rupture is the mechanical failure of the heterogeneous fibrous plaque tissue. Local characterization of the tissue's failure properties and the collagen architecture are of great importance to have insights in plaque rupture for clinical event prevention. Previous studies were limited to average rupture properties and global structural characterization, and did not provide the necessary local information. In this study, we assessed the local collagen architecture and failure properties of fibrous plaque tissue, by analyzing 30 tissue strips from 18 carotid plaques. Our study framework entailed second harmonic generation imaging for local collagen orientation and dispersion, and uniaxial tensile testing and digital image correlation for local tissue mechanics. The results showed that 87% of the imaged locations had collagen orientation close to the circumferential direction (0°) of the artery, and substantial dispersion locally. All regions combined, median [Q1:Q3] of the predominant angle measurements was -2° [-16°:16°]. The stretch ratio measurements clearly demonstrated a nonuniform stretch ratio distribution in the tissue under uniaxial loading. The rupture initiation regions had significantly higher stretch ratios (1.26 [1.15-1.40]) than the tissue average stretch ratio (1.11 [1.10-1.16]). No significant difference in collagen direction and dispersion was identified between the rupture regions and the rest of the tissue. The presented study forms an initial step towards gaining better insights into the characterization of local structural and mechanical fingerprints of fibrous plaque tissue in order to aid improved assessment of plaque rupture risk. Statement of significance: Plaque rupture risk assessment, critical to prevent cardiovascular events, requires knowledge on local failure properties and structure of collagenous plaque tissue. Our current knowledge is unfortunately limited to tissue's overall ultimate failure properties with scarce information on collagen architecture. In this study, local failure properties and collagen architecture of fibrous plaque tissue were obtained. We found predominant circumferential alignment of collagen fibers with substantial local dispersion. The tissue showed nonuniform stretch distribution under uniaxial tensile loading, with high stretches at rupture spots. This study highlights the significance of local mechanical and structural assessment for better insights into plaque rupture and the potential use of local stretches as risk marker for plaque rupture for patient-specific clinical applications.

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.

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.