The effect of presence and location of microcalcifications on atherosclerotic plaque rupture: A tissue-engineering approach

Rupture of the cap of an atherosclerotic plaque can trigger thrombotic cardiovascular events. It has been suggested, through computational models, that the presence and specific location of microcalcifications in the atherosclerotic cap can increase the risk of cap rupture. However, the experimental confirmation of this hypothesis is lacking. In this study, we investigated how the presence and location of microcalcifications, relative to the lumen, influence (local) mechanics and rupture behavior of atherosclerotic plaque caps. Using tissue-engineered fibrous cap analogs with hydroxyapatite (HA) clusters to mimic calcifications in human plaque caps, we replicated the microcalcification distribution observed in human carotid plaques, as identified by our histological analysis. The analogs were imaged using multiphoton microscopy with second-harmonic generation to assess local collagen fiber orientation and dispersion. Subsequently, they underwent uniaxial tensile testing to failure, during which local strain and failure characteristics were analyzed. Our results revealed that HA clusters, particularly those in the luminal region, contribute to increased local collagen fiber dispersion. Moreover, the presence of HA clusters reduced both failure tensile stress and strain in the TE cap analogs. Besides, the rupture location shifted toward the site of HA clusters. Additionally, rupture initiation was consistently found in high-strain regions, and in 86 % of the analogs, even at the highest strain location in the sample. Our findings suggest that microcalcification clusters in plaque caps may increase the cap rupture risk and relocate the rupture site. Moreover, local strain measurements can serve as an additional tool for plaque cap rupture risk assessment.