Rupture of atherosclerotic plaque caps is the cause of many disabling or lethal cardiovascular events, such as stroke and myocardial infarction. Microcalcifications (<50 µm) have been shown, in computational models, to affect the biomechanical stability of the cap. The current study aims to develop a tissue-engineered model of the atherosclerotic fibrous cap with microcalcifications produced by mesenchymal stromal cells (MSCs). Human MSCs are seeded in fibrin gels and cultured for 2 weeks in medium supplemented with TGF-β1 to induce smooth muscle cell differentiation and collagenous matrix formation. Afterward, mineralizing medium stimulates microcalcification formation for an additional 4 weeks. Tissue-engineered structures are imaged after culture with second harmonic generation microscopy with a hydroxyapatite probe, showing collagenous matrix with microcalcifications. Mechanical characterization shows the effect of microcalcifications on global tissue mechanics, as the ultimate stress at rupture of the tissue is significantly lower compared to control tissues. The amount of calcification, determined by histological analysis, is correlated to the decrease in ultimate tensile stress, with a higher amount of microcalcification resulting in weakened mechanical properties. The developed tissue-engineered plaque cap model with biologically formed collagenous matrix and microcalcifications offers valuable insight into the impact of microcalcifications on biomechanical stability.

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.

Atherosclerotic plaque rupture can lead to thrombotic cardiovascular events such as stroke and myocardial infarction. Computational models have shown that microcalcifications (calcified particles with a diameter < 50 μm) in the atherosclerotic plaque cap can increase cap tissue stresses and consequently contribute to plaque rupture. Microcalcification characteristics, such as particle size and volume fraction, have been implicated to affect cap stresses. However, the effect of these characteristics on tissue mechanics within a collagenous matrix, has not been investigated experimentally. In this study, we employ a tissue-engineered model of the atherosclerotic plaque cap with human myofibroblasts to assess the impact of microcalcification size and volume fraction on cap mechanics and rupture. To mimic human microcalcification size and volume, hydroxyapatite microparticles, in two size ranges (diameter up to 5 μm or up to 50 μm) and two volumes (1 v/v% and 5 v/v%) were incorporated homogenously throughout the tissue-engineered model. 5 v/v% of particles caused a significant lowering of the mechanical properties as was shown by a decrease in stiffness and ultimate tensile stress under uniaxial tensile loading. Additionally, the 5 v/v% of hydroxyapatite particles, in both size ranges, caused a reduced tissue compaction during culture. This might indicate that hydroxyapatite particles influence mechanobiological processes governing tissue organisation and consequent tissue mechanics. These experimental data support computational findings regarding the detrimental role of microcalcifications on cap rupture risk and highlight the importance of volume fraction. Furthermore, this study indicates an additional importance to look at the interplay between calcification, its effect on plaque cap-resident cells and the consequent effect on tissue mechanics.