Imaging and analysis of microcalcifications and lipid/necrotic core calcification in fibrous cap atheroma.

The presence of microcalcifications (µCalcs) >5 µm within the cap of human fibroatheroma has been shown to produce a 200-700% increase in peak circumferential stress, which can transform a stable plaque into a vulnerable one, whereas µCalcs < 5 µm do not appear to increase risk. We quantitatively examine the possibility to distinguish caps with µCalcs > 5 µm based on the gross morphological features of fibroatheromas, and the correlation between the size and distribution of µCalcs in the cap and the calcification in the lipid/necrotic core beneath it. Atherosclerotic lesions (N = 72) were imaged using HR-µCT at 2.1-µm resolution for detailed analysis of atheroma morphology and composition, and validated using non-decalcified histology. At 2.1-µm resolution one observes four different patterns of calcification within the lipid/necrotic core, and is able to elucidate the 3D spatial progression of the calcification process using these four patterns. Of the gross morphological features identified, only minimum cap thickness positively correlated with the existence of µCalcs > 5 µm in the cap. We also show that µCalcs in the cap accumulate in the vicinity of the lipid/necrotic core boundary with few on the lumen side of the cap. HR-µCT enables three-dimensional assessment of soft tissue composition, lipid content, calcification patterns within lipid/necrotic cores and analysis of the axial progression of calcification within individual atheroma. The distribution of µCalcs within the cap is highly non-uniform and decreases sharply as one proceeds from the lipid pool/necrotic core boundary to the lumen.

Effect of tissue properties, shape and orientation of microcalcifications on vulnerable cap stability using different hyperelastic constitutive models.

Approximately half of all cardiovascular deaths associated with acute coronary syndrome occur when the thin fibrous cap tissue overlying the necrotic core in a coronary vessel is torn, ripped or fissured under the action of high blood pressure. From a biomechanics point of view, the rupture of an atheroma is due to increased mechanical stresses in the lesion, in which the ultimate stress (i.e. peak circumferential stress (PCS) at failure) of the tissue is exceeded. Several factors including the cap thickness, morphology, residual stresses and tissue composition of the atheroma have been shown to affect the PCS. Also important, we recently demonstrated that microcalcifications (µCalcs>5 µm are a common feature in human atheroma caps, which behave as local stress concentrators, increasing the local tissue stress by at least a factor of two surpassing the ultimate stress threshold for cap tissue rupture. In the present study, we used both idealized µCalcs with spherical shape and actual µCalcs from human coronary atherosclerotic caps, to determine their effect on increasing the circumferential stress in the fibroatheroma cap using different hyperelastic constitutive models. We have found that the stress concentration factor (SCF) produced by µCalcs in the fibroatheroma cap is affected by the material tissue properties, µCalcs spacing, aspect ratio and their alignment relative to the tensile axis of the cap.

Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries.

Using 2.1-µm high-resolution microcomputed tomography, we have examined the spatial distribution, clustering, and shape of nearly 35,000 microcalcifications (µCalcs) ≥ 5 µm in the fibrous caps of 22 nonruptured human atherosclerotic plaques. The vast majority of these µCalcs were <15 µm and invisible at the previously used 6.7-µm resolution. A greatly simplified 3D finite element analysis has made it possible to quickly analyze which of these thousands of minute inclusions are potentially dangerous. We show that the enhancement of the local tissue stress caused by particle clustering increases rapidly for gap between particle pairs (h)/particle diameter (D) < 0.4 if particles are oriented along the tensile axis of the cap. Of the thousands of µCalcs observed, there were 193 particle pairs with h/D ≤ 2 (tissue stress factor > 2), but only 3 of these pairs had h/D ≤ 0.4, where the local tissue stress could increase a factor > 5. Using nondecalcified histology, we also show that nearly all caps have µCalcs between 0.5 and 5 µm and that the µCalcs ≥ 5 µm observed in high-resolution microcomputed tomography are agglomerations of smaller calcified matrix vesicles. µCalcs < 5 µm are predicted to be not harmful, because the tiny voids associated with these very small particles will not explosively grow under tensile forces because of their large surface energy. These observations strongly support the hypothesis that nearly all fibrous caps have µCalcs, but only a small subset has the potential for rupture.

The explosive growth of small voids in vulnerable cap rupture; cavitation and interfacial debonding.

While it is generally accepted that ruptures in fibrous cap atheromas cause most acute coronary deaths, and that plaque rupture occurs in the fibrous cap at the location where the tissue stress exceeds a certain critical peak circumferential stress, the exact mechanism of rupture initiation remains unclear. We recently reported the presence of multiple microcalcifications (µCalcs) <50 µm diameter embedded within the fibrous cap, µCalcs that could greatly increase cap instability by introducing up to a 5-fold increase in local tissue stress. Here, we explore the hypothesis that, aside from cap thickness, µCalc size and interparticle spacing are principal determinants of cap rupture risk. Also, we propose that cap rupture is initiated near the poles of the µCalcs due to the presence of tiny voids that explosively grow at a critical tissue stress and then propagate across the fibrous cap. We develop a theoretical model based on classic studies in polymeric materials by Gent (1980), which indicates that cavitation as opposed to interfacial debonding is the more likely mechanism for cap rupture produced by µCalcs <65 µm diameter. This analysis suggests that there is a critical µCalc size range, from 5 µm to 65 µm, in which cavitation should be prevalent. This hypothesis for cap rupture is strongly supported by our latest high resolution µCT studies in which we have observed trapped voids in the vicinity of µCalcs within fibrous caps in human coronaries.

A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture.

The role of microcalcifications (µCalcs) in the biomechanics of vulnerable plaque rupture is examined. Our laboratory previously proposed (Ref. 44), using a very limited tissue sample, that µCalcs embedded in the fibrous cap proper could significantly increase cap instability. This study has been greatly expanded. Ninety-two human coronary arteries containing 62 fibroatheroma were examined using high-resolution microcomputed tomography at 6.7-µm resolution and undecalcified histology with special emphasis on calcified particles <50 µm in diameter. Our results reveal the presence of thousands of µCalcs, the vast majority in lipid pools where they are not dangerous. However, 81 µCalcs were also observed in the fibrous caps of nine of the fibroatheroma. All 81 of these µCalcs were analyzed using three-dimensional finite-element analysis, and the results were used to develop important new clinical criteria for cap stability. These criteria include variation of the Young's modulus of the µCalc and surrounding tissue, µCalc size, and clustering. We found that local tissue stress could be increased fivefold when µCalcs were closely spaced, and the peak circumferential stress in the thinnest nonruptured cap (66 µm) if no µCalcs were present was only 107 kPa, far less than the proposed minimum rupture threshold of 300 kPa. These results and histology suggest that there are numerous µCalcs < 15 µm in the caps, not visible at 6.7-µm resolution, and that our failure to find any nonruptured caps between 30 and 66 µm is a strong indication that many of these caps contained µCalcs.