A primary inflammatory malignant fibrous histiocytoma of the kidney is reported. The differential diagnosis from other sarcomas and pseudosarcomatous lesions is discussed on the grounds of the histologic, immunohistochemical and ultrastructural results. The neoplasm was made up of histiocytes, fibroblasts, myofibroblasts, foam cells and undifferentiated mesenchymal cells with admixed granulocytes, lymphocytes and plasma cells. The immunohistochemical study showed a positivity for alpha-1-antichymotrypsin and a weak positivity for alpha-1-antitrypsin in mononuclear and pleomorphic multinucleated tumor cells. PASpositive, diastase-resistant intracytoplasmic hyaline globules in necrotic cells, examined by electron microscopy, most likely represent lysosomal structures, in accordance with the theories of De Duve, Vattiaux and Von Ardenne.
Abstract Mimicking the properties of the extracellular matrix is crucial for developing in vitro models of the physiological microenvironment of living cells. Among other techniques, 3D direct laser writing (DLW) has emerged as a promising technology for realizing tailored 3D scaffolds for cell biology studies. Here, results based on DLW addressing basic biological issues, e.g., cell‐force measurements and selective 3D cell spreading on functionalized structures are reviewed. Continuous future progress in DLW materials engineering and innovative approaches for scaffold fabrication will enable further applications of DLW in applied biomedical research and tissue engineering.
Abstract Background Coronary calcification is associated with high risk for cardiovascular events. However, its impact on plaque vulnerability is incompletely understood. In the present study we defined the intrinsic calcification angle (ICA) as the angle externally projected by a vascular calcification and analyzed its role as novel feature of coronary plaque vulnerability in patients with type 2 diabetes. Methods Optical coherence tomography was used to determine ICA in 219 calcifications from 56 patients with stable coronary artery disease (CAD) and 143 calcifications from 36 patients with acute coronary syndrome (ACS). We then used finite elements analysis to gain mechanistic insight into the effects of ICA. Results Minimal (139.8 ± 32.8° vs. 165.6 ± 21.6°, p < 0.001) and mean ICA (164.1 ± 14.3° vs. 176.0 ± 8.4°, p < 0.001) were lower in ACS vs. stable CAD patients. Mean ICA predicted ACS with very good diagnostic efficiency (AUC = 0.840, 95% CI 0.797–0.882, p < 0.001, optimal cut-off 175.9°); younger age (OR 0.95 per year, 95% CI 0.92–0.98, p = 0.002), male sex (OR 2.18, 95% CI 1.41–3.38, p < 0.001), lower HDL-cholesterol (OR 0.82 per 10 mg/dl, 95% CI 0.68–0.98, p = 0.029) and ACS (OR 14.71, 95% CI 8.47–25.64, p < 0.001) were determinants of ICA < 175.9°. A lower ICA predicted ACS (OR for 10°-variation 0.25, 95% CI 0.13–0.52, p < 0.001) independently from fibrous cap thickness, presence of macrophages or extension of lipid core. In finite elements analysis we confirmed that lower ICA causes increased stress on a lesion’s fibrous cap; this effect was potentiated in more superficial calcifications and adds to the destabilizing role of smaller calcifications. Conclusion Our clinical and mechanistic data for the first time identify ICA as a novel feature of coronary plaque vulnerability.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Plaque rupture occurs if stress within coronary lesions exceeds the protection exerted by the fibrous cap overlying the necrotic lipid core. However, very little is known about the biomechanical stress exerting this disrupting force. Employing optical coherence tomography (OCT), we generated plaque models and performed finite-element analysis to simulate stress distributions within the vessel wall in 10 ruptured and 10 non-ruptured lesions. In ruptured lesions, maximal stress within fibrous cap (peak cap stress [PCS]: 174 ± 67 vs. 52 ± 42 kPa, p<0.001) and vessel wall (maximal plaque stress [MPS]: 399 ± 233 vs. 90 ± 95 kPa, p=0.001) were significantly higher compared to non-ruptured plaques. Ruptures arose in the immediate proximity of maximal stress concentrations (angular distances: 21.8 ± 30.3° for PCS vs. 20.7 ± 23.7° for MPS); stress concentrations excellently predicted plaque rupture (area under the curve: 0.940 for PCS, 0.950 for MPS). This prediction of plaque rupture was superior to established vulnerability features such as fibrous cap thickness or macrophage infiltration. In conclusion, OCT-based finite-element analysis effectively assesses plaque biomechanics, which in turn predicts plaque rupture in patients. This highlights the importance of morpho-mechanic analysis assessing the disrupting effects of plaque stress. eLife digest Heart attacks are caused by a blockage in arteries that supply oxygen to the heart. This often happens when fatty deposits (or ‘plaques’) that line blood vessels break off and create a clot. To identify individuals most at risk of this occurring, physicians currently use symptoms, family history, blood tests, imaging and surgical procedures. But better methods are needed. Imaging blockages in the arteries of individuals who died from heart attacks highlighted certain plaque characteristics that increase the risk of a rupture. Further understanding the forces that lead to these fatty deposits breaking off may help scientists to develop improved heart attack prediction methods. Using patient-specific computer simulations, Milzi et al. show it is possible to predict where plaques are most likely to rupture in an individual, based on biomechanical stresses on the deposits in the artery. The models also showed how forces on the external layers of the plaque played a pivotal role in breakages. More research is needed to confirm the results of this study and to develop automated ways for measuring the stress exerted on plaques in the arteries. If that research is successful, biomechanical analyses of artery plaques in routine patient assessments may one day allow physicians to predict heart attacks and provide life-saving preventive care. Introduction Coronary artery disease (CAD) is one of the major causes of morbidity and mortality in the Western World (Center for Disease Control, 2013). One of the main challenges in modern therapy of patients with CAD is the detection of atherosclerotic plaques that do not yet limit flow in the coronary artery, but may potentially cause an acute coronary syndrome by rupturing with subsequent acute occlusion of the artery. Decades ago, these lesion entities have been defined as ‘vulnerable plaques’ (Virmani et al., 2003). Pathology-based studies (Virmani et al., 2003; Falk, 1989; Burke et al., 1997; Virmani et al., 2000) as well as intravascular imaging (Kato et al., 2012; Kubo et al., 2013; Reith et al., 2014; Uemura et al., 2012; Ehara et al., 2004) have been used over time to explore potential features of plaque vulnerability, that is morphological characteristics which may predispose a plaque to rupture. The presently accepted features assessed using intravascular imaging include the thickness of the fibrous cap (FCT) (Kato et al., 2012; Kubo et al., 2013; Reith et al., 2014; Uemura et al., 2012), the extent of the necrotic lipid core (Kato et al., 2012), the presence of macrophages (Kato et al., 2012; Reith et al., 2014; Uemura et al., 2012), microvessels (Kato et al., 2012; Uemura et al., 2012), small calcifications (Ehara et al., 2004; Reith et al., 2018), or a positive remodeling (Varnava et al., 2002). Features of coronary plaque vulnerability may also be assessed with different imaging modalities, such as coronary computed tomography angiography, and include also low-attenuation plaques and a higher plaque burden (Conte et al., 2017). These features may predict the incidence of future major cardiac events (Conte et al., 2017; Prati et al., 2020), if such high-risk plaques are not timely recognized and sealed (Dettori et al., 2020). From a mechanistic perspective, plaque rupture originates from an excessive stress concentration on the fibrous cap, which trespasses the tensile strength of the material and ultimately causes material failure and consequently plaque rupture. The fibrous cap yields, in this view, a protective effect, by avoiding the exposure of prothrombotic material present in the necrotic lipid core. Thus, it is not surprising that minimal FCT has been considered the most established parameter of plaque vulnerability (Kato et al., 2012; Kubo et al., 2013; Reith et al., 2014; Uemura et al., 2012): in fact, the thickness of the fibrous cap seems a good surrogate parameter of its ability to resist stresses. Still, evaluating solely the fibrous cap addresses only the main stabilizing factor in plaque biomechanics, leaving the destabilizing factors almost completely ignored. However, plaque rupture occurs if the stress within coronary lesions exceeds the protection exerted by the fibrous cap. Therefore, it is of utmost importance to also calculate the stress within the plaque as the disrupting force within the lesion and not only focus on the protective effects of the fibrous cap. However, very little is known about the determinants of the disrupting force pushing coronary plaques to rupture. In addition, the spatial relationship between rupture and the balance of stabilizing and destabilizing forces in the plaque remains largely unexplored: in other words, whether plaque rupture occurs at the site with the lowest FCT, as point of least resistance, or rather in sites of highest stress concentration, as point of maximal disruption is unclear. It is self-evident that such research questions cannot be addressed by ‘standard’ plaque imaging analysis. Therefore, we chose to develop a morpho-metric finite-element analysis to determine the influence of plaque morphology on the biomechanics of coronary lesions in patients and investigate the relationship between this calculated morpho-mechanic stress and plaque rupture. As a source of patient-specific, high-quality images of coronary plaques for this proof-of-concept study, we selected optical coherence tomography (OCT), an established intravascular imaging technique which, due to a supreme resolution (up to 10–20 µm), is able to depict peri-luminal structures with high accuracy (Burgmaier et al., 2014). Based on the images obtained, we generated patient-specific reconstructions of coronary plaques, which we used as a basis to analyze stress concentration as a possible predictor of plaque rupture. Results Population and lesion characteristics Of the 20 patients with type 2 diabetes enrolled in this study, 10 underwent coronary angiography due to acute coronary syndrome and 10 due to chronic coronary syndrome. All selected patients with acute coronary syndromes showed plaque rupture as the morphological correlate. Patients with (n = 10) and without (n = 10) plaque rupture did not differ with respect to their clinical characteristics, apart from a worse glycemic control in patients with plaque rupture (HbA1c: 7.4 ± 1.4 vs. 6.1 ± 0.5, p=0.026). As expected, ruptured lesions presented a lower FCT (minimal FCT: 49 ± 10 µm vs. 97 ± 15 µm, p<0.001; mean FCT: 94 ± 17 µm vs. 133 ± 12 µm, p=0.006), a more extensive necrotic lipid core (lipid arc: 178 ± 39° vs. 110 ± 8°, p=0.001; lipid volume index: 9876 ± 3088 vs. 3853 ± 1294, p=0.011), and a higher incidence of thin-capped fibroatheromas (70% vs. 0%, p=0.003) compared to non-ruptured lesions. Patients and lesions characteristics are reported in Table 1. Table 1 Patients and lesions characteristics. Abbreviations: BMI = body mass index, FCT = fibrous cap thickness, TCFA = thin-capped fibroatheroma. Non-rupturedRupturedpn = 10n = 10Clinical characteristicsMale sex (n, %)8 (80%)8 (80%)1.000Age (years)68 ± 770 ± 100.689Hypertension (n, %)10 (100%)9 (90%)0.305Hyperlipidemia (n, %)7 (70%)6 (60%)0.639Nicotine use (n, %)3 (30%)4 (40%)0.639BMI (kg/m2)33 ± 530 ± 70.230Total cholesterol (mg/dl)172 ± 43165 ± 330.663LDL-c (mg/dl)106 ± 41101 ± 320.724HDL-c (mg/dl)39 ± 639 ± 60.972Triglycerides (mg/dl)161 ± 62180 ± 1080.640HbA1c (%)6.1 ± 0.57.4 ± 1.40.026hsCRP5.0 ± 2.326.7 ± 46.90.175Aspirine therapy (n, %)10 (100%)9 (90%)0.305Statine therapy (n, %)6 (60%)6 (60%)1.000Lesion characteristicsMinimal FCT (µm)97 ± 1549 ± 10<0.001Mean FCT (µm)133 ± 1294 ± 170.006Maximal lipid arc (°)110 ± 8178 ± 390.001Lipid volume arc (mm*°)3853 ± 12949876 ± 30880.011Presence of TCFA (n, %)0 (0%)7 (70%)0.003Presence of macrophages (n, %)4 (40%)6 (60%)0.371Presence of spotty calcifications (n, %)7 (70%)8 (80%)0.527 Stress analysis in ruptured and non-ruptured plaques In ruptured plaques, the maximal stress within the fibrous cap (peak cap stress) was significantly higher than in non-ruptured ones (174 ± 67 vs. 52 ± 42 kPa, p<0.001). Furthermore, also the maximal stress within the whole plaque (maximal plaque stress) was more than fourfold higher in ruptured plaques compared to stable ones (399 ± 233 vs. 90 ± 95 kPa, p=0.001). Exemplary images of stress concentrations on the fibrous cap and overall in plaques, as well as a box-plot depicting stress concentrations in lesions with and without rupture are shown in Figure 1. Variation of stress distribution in dependence of various model assumptions are reported in the Supplementary Results in Appendix 1. Figure 1 Download asset Open asset Higher stress concentrations in ruptured plaques than in non-ruptured coronary plaques. In (A, D), model reconstructions for ruptured and non-ruptured plaques are shown. In both fibrous cap (B) vs. (E) and vessel wall (C) vs. (F), ruptured plaques present higher stress concentrations than non-ruptured ones. Color-code scale is the same for both reconstructions, and maximal stress is shown at the upper edge of the scale. In (G) is shown a box-plot demonstrating the different stress concentrations both on the fibrous cap (in blue) and in the whole plaque (in red). Spatial correlation between stress concentrations and rupture site in OCT images After documenting much greater stress in ruptured compared with stable plaques, we assessed the effects of these morphology-driven peaks of intra-plaque stress on the lesion and if points of highest stress co-localize with plaque rupture within coronary lesions. Thus, we aimed to analyze the spatial correlation between the rupture point, as visualized in OCT images, and the point of maximal stress concentration, in order to further support the mechanistic role of stress concentrations in the fibrous cap in the genesis of plaque rupture. Only 50% of the plaque ruptures were in the shoulder region. The angle between the peak cap stress and the detectable rupture site was very low at 21.8 ± 30.3°; the angle between the maximal plaque stress and the detectable rupture site was even lower (20.7 ± 23.7°). Overall, 50% of plaque ruptures occurred within 10° of the peak cap stress and of the maximal plaque stress, suggesting that plaque rupture occurs in the very proximity of highest stress concentrations. A graphical presentation of spatial correlation between the sites presenting the peak cap stress and the maximal plaque stress in finite-element analysis and the rupture site in OCT pullback is shown in Figure 2. Figure 2 Download asset Open asset Coronary plaques rupture in the proximity of stress concentrations. In (A, B), stress concentrations on the fibrous cap and on the vessel wall in the same coronary segment; in (C), the points of maximal stress concentration on the fibrous cap (blue asterisk) and on the vessel wall (red hash) are reported, in the OCT-frame immediately following the one used for reconstruction shown. Here, the rupture is marked with a white arrow. In (D, E), polar histograms showing distribution of angles between rupture and maximal stress on fibrous cap (D) and on the vessel wall (E). Angles higher than 120° have not been detected and for this reason are not shown in the polar graphs. Diagnostic value of stress distribution in predicting plaque rupture In order to assess the diagnostic efficacy of finite-element analyses in predicting plaque rupture, we performed receiver operating characteristic (ROC) analysis for both peak cap stress and maximal plaque stress. In our cohort, we could demonstrate that both peak cap stress (area under the curve [AUC] 0.940) and maximal plaque stress (AUC 0.950) predict plaque rupture with excellent accuracy. Optimal cut-off for prediction of plaque rupture were 150.5 kPa (Sensitivity 90%; Specificity 80%) for peak cap stress and 169.5 kPa (Sensitivity 90%; Specificity 90%) for maximal plaque stress. ROC curves for the prediction of plaque rupture are shown in Figure 3. Figure 3 Download asset Open asset Stress concentration predicts plaque rupture with excellent diagnostic efficiency. ROC curves for the prediction of plaque rupture are shown for maximal stress in the fibrous cap (A) and in the vessel wall (B) of simulated vessels. To assess the possible additional value of finite-elements analysis compared to known features of plaque vulnerability, we performed ROC analysis for prediction of plaque rupture. Minimal FCT (AUC 0.630) and mean FCT (AUC 0.630) presented sufficient diagnostic accuracy; lipid volume index (AUC 0.870) showed a very good diagnostic accuracy in predicting plaque rupture. Both peak cap stress and maximal plaque stress presented a significantly superior diagnostic efficiency in predicting plaque rupture compared to mean FCT (p=0.027 vs. peak cap stress; p=0.036 vs. maximal plaque stress), minimal FCT (p vs. peak cap stress: 0.027; p vs. maximal plaque stress = 0.036) and extent of macrophage infiltration (p=0.003 vs. peak cap stress; p=0.001 vs. maximal plaque stress). Furthermore, diagnostic efficiency of both peak cap stress and maximal plaque stress was numerically superior (albeit non-significant) compared to that of lipid volume index (p=0.409 vs. peak cap stress and p=0.386 vs. mean plaque stress). We combined OCT-derived morphologic features of plaque vulnerability by calculating two previously validated scores, shown in the CLIMA study (Prati et al., 2020) and in a publication by Burgmaier et al., 2014. Both scores could predict plaque rupture with very good to excellent diagnostic efficiency (AUC for CLIMA score: 0.870; AUC for Burgmaier score: 0.900), which however remained numerically lower than results of our morpho-mechanic analysis. ROC curves, including comparison with results of finite-element analyses, are shown in Figure 4. Figure 4 Download asset Open asset Comparison of stress concentration to ‘classical’ features of plaque vulnerability in prediction of plaque rupture. ROC curves for the prediction of plaque rupture for minimal FCT (A), mean FCT (B), lipid volume index (C), and macrophage volume index (D). In (E), ROC curves for two established OCT scores combining different features of plaque vulnerability are depicted (respectively: scores calculated according to Burgmaier et al., 2014 in blue and according to the CLIMA study (Prati et al., 2020 in grey) and compared to the ROC curves for peak cap stress (PCS, black dotted line) or maximal plaque stress (MPS, red dashed line). Discussion The main findings of our work are: OCT-based morpho-mechanic finite-element analysis is a feasible tool to obtain patient-specific assessment of biomechanics in coronary lesions; our model detects significantly greater stress concentrations (more than threefold for peak cap stress and more than fourfold for maximal plaque stress) in ruptured plaques compared to non-ruptured plaques and predicts plaque rupture with excellent diagnostic efficiency; plaque rupture arises in the proximity of stress concentration areas; the diagnostic efficiency of our model was superior to established morphologic features of plaque vulnerability, as for instance the thickness of the fibrous cap, the extent of the necrotic lipid core, and the extent of plaque macrophage infiltration. Current research on coronary plaque vulnerability mainly focused on morphological features of vulnerable plaque. The main morphologic characteristic that emerged from previous studies is FCT (Kato et al., 2012; Kubo et al., 2013; Reith et al., 2014; Uemura et al., 2012), which has been constantly related to a higher incidence of plaque rupture. This is not surprising, considering its role as the last barrier resisting the stresses exerted on the atheroma and thus preventing plaque rupture. Measuring FCT, in this perspective, is indirectly assessing the mechanical resistance – and thus the protection – of the coronary plaque to rupture. But the decrease of this protection in cases of a low FCT cannot completely determine plaque rupture, which arises from the difference between protection exerted by the fibrous cap and its disrupting forces. Data on these disrupting forces as the primum movens of plaque rupture are still very scarce (Loree et al., 1992; Cheng et al., 1993; Finet et al., 2004). Therefore, in this proof-of-concept study, we aimed to assess stress concentrations on and determined by coronary plaques in vivo and analyzed their relationship with plaque rupture. To perform this analysis, we developed a morpho-mechanic model reconstructed from in vivo OCT images. Previous pioneering studies could show the applicability of finite-element analysis to the assessment of the biomechanics of the atherosclerotic plaque (Loree et al., 1992; Cheng et al., 1993). However, pathology-based simulations lack a direct applicability in patient care. Other studies, although based on intravascular imaging, were based on single lesions, which were analyzed on a qualitative basis (Finet et al., 2004; Reith et al., 2019). On the contrary, our study is to the best of our knowledge first to perform complex mechanistic analysis of coronary plaques based on high-definition intravascular imaging on a patient-specific level, with a high potential for direct clinical application. Stress concentration is closely associated to plaque rupture First, we could in vivo detect higher stress concentrations both within the fibrous cap and within the whole plaque in ruptured plaques compared to non-ruptured plaques. This is in line with previous simulation studies based on pathology specimens (Loree et al., 1992; Cheng et al., 1993) and confirms our hypothesis of stress concentration as a central factor in the genesis of plaque rupture. As a further step in confirming this theory, we extended current knowledge by showing that plaque ruptures arise in the immediate proximity of maximal stress concentrations. This spatial coincidence between maximal stress concentrations and sites of plaque rupture confirms the causal link between plaque biomechanics on the one hand and plaque rupture and acute coronary syndromes on the other. In fact, on the basis of our data, it is tempting to speculate about the mechanistic process eventually leading to plaque rupture. Stress within the fibrous cap and overall within the plaque is concentrated in a pattern which is closely dependent on the morphology of the plaque (for instance, in dependence of the extent and morphology of the necrotic lipid core and/or of calcification). A specific force is exerted on each point of the fibrous cap; should this stress concentration trespass the ultimate tensile strength of the vessel wall – which is highly likely to happen in points of maximal stress concentration – material failure and, eventually, plaque rupture occurs. An interesting question is, of course, the magnitude of the threshold that needs to be trespassed to cause material failure and plaque rupture. A previous pathology-based study by Cheng et al., 1993 sets the stress threshold for plaque rupture to 300 kPa. In our study, we found 150.5 kPa in the fibrous cap and 169.5 kPa in the vessel wall to be the optimal thresholds for predicting plaque rupture. The difference between these values and the initially estimated threshold of 300 kPa may be explained through the different imaging modalities used to reconstruct the coronary plaque. In fact, in pathology specimens (on which the 300 kPa value is based on), the fixation process may cause a shrinkage of the fibrous cap with a shortening of ca. 10–20% compared to the thickness measured in vivo with OCT. This effect may be sufficient to explain the numerical difference of the ‘critical’ peak cap stress needed for plaque rupture, especially considering – as pointed out by Finet et al. – that the relationship between FCT and peak cap stress is exponential (Finet et al., 2004). Further clinical validation of these thresholds is, however, needed. Another possible explanation might be the hyperelastic behavior of certain plaque components, which has been described by some authors previously (Yang et al., 2009; Cardoso et al., 2014); such an effect is, however, only present at large displacements (>20–30% of the initial dimensions) (Yang et al., 2009; Kobielarz et al., 2020), which are way over the average displacements reported in our study. In this range, a linear equation adequately depicts the behavior of the material. Also, stress on the lesion in patients is not only derived from the static factors included in our analysis, but also dynamic flow shear stress caused by blood flow (Bourantas et al., 2020), which was not included in our study. Potential clinical utility of finite-element analysis in predicting plaque rupture Although dynamic flow shear stress was not included in our analysis, we could also show an excellent diagnostic efficiency of our finite-element analysis in predicting plaque rupture. Furthermore, the model we developed presented a clearly superior diagnostic efficiency compared with accepted parameters of plaque vulnerability as FCT (Kato et al., 2012; Kubo et al., 2013; Reith et al., 2014; Uemura et al., 2012) or plaque macrophage infiltration (Kato et al., 2012; Reith et al., 2014; Uemura et al., 2012) and a numerically superior efficiency when compared to lipid volume index (Kato et al., 2012) in our cohort. The diagnostic efficiency of our model even presented a comparable efficiency when compared with the combination of lesion morphologies including FCT, plaque macrophage infiltration, and lipid volume index. In the light of these findings, it may be tempting to speculate about the strong link existing between stress concentrations and plaque rupture, which may explain the excellent predictive value of our model and may pave the way for widespread use of morpho-mechanical analysis in the clinical routine to detect vulnerable plaques. Limitations To the best of our knowledge, although we are first to develop an OCT-based finite-element model allowing patient-specific analysis of plaque biomechanics, several limitations have to be acknowledged. First of all, we faithfully reproduced plaque morphology in a 2D-based reconstruction; on the other hand, we are not taking into account several phenomena that may influence stress concentration, as for instance the longitudinal structure of the plaque or the longitudinal profile of the stenosis and flow shear stress – the relevance of these factors needs to be addressed in future studies. Furthermore, in spite of the excellent resolution of OCT in the near field, we cannot exclude imprecisions in the segmentation due to limited light penetration to the deeper vessel wall. Limited tissue penetration, in fact, does not allow to assess sites with positive remodeling, which may yield higher stresses due to accelerated plaque growth and therefore cause a higher rupture risk. This needs to be assessed through different imaging modalities, such as IVUS or coronary computed tomography. Moreover, dedicated computational techniques in order to reconstruct the deep structure of the lipid core (Kok et al., 2016) have not been employed, in order to simplify plaque reconstruction; moderate imprecisions in the deep contour of the lipid core cannot therefore be excluded. For our study, we employed a linear elastic model; other authors, though, suggest an hyperelastic behavior of the vessel wall, particularly for displacements > 20% of the initial dimensions (Yang et al., 2009; Kobielarz et al., 2020). In spite of an average displacement ‘small enough’ (10%) to justify linearity, we cannot exclude underestimation of stress concentrations for very few plaques with larger displacements. To the best of our knowledge, although being the first to employ a morpho-metric approach in assessing plaque vulnerability on in vivo intravascular imaging, this pilot study still includes a low number of patients chosen among patients with and without plaque rupture, which may reduce the reliability of exact AUC and cut-off values in our predictive models; further analyses are needed to confirm the results of our proof-of-concept analysis. Materials and methods Patient population Request a detailed protocol We retrospectively selected 20 patients with type 2 diabetes mellitus and CAD, who underwent OCT prior to percutaneous coronary intervention at the Department of Cardiology of the University Hospital of the RWTH Aachen. Clinical presentation was stable CAD without evidence of plaque rupture in the OCT pullback (n = 10) or acute coronary syndrome with plaque rupture (n = 10). Sample size calculation was performed based on previous results of calculation of mechanical stresses in histopathological samples, resulting in a minimal sample size of seven lesions per group in order to achieve α = 0.001 and power of 0.95; this was then arbitrarily rounded to 10 lesions per group. Informed consent of all patients was obtained prior to inclusion in the study. The study was approved by the Ethics Committee of the University Hospital of the RWTH Aachen (EK 071/11 and EK 277/12) and is in accordance with the declaration of Helsinki on ethical principles for medical research involving human subjects. OCT image acquisition and analysis Request a detailed protocol The acquisition of OCT pullbacks was performed as previously described in the literature (Tearney et al., 2012; Milzi et al., 2017). In brief, OCT images were acquired prior to coronary intervention using a frequency domain OCT C7XR system and the DragonFly catheter (St. Jude Medical Systems; Lightlab Imaging Inc, Westford, MA). Blood removal was obtained by the injection of 14 ml contrast dye (iodixanol) at a flow rate of 4 ml/s through the guiding catheter. Image acquisition was obtained with automated pull-back rate of 20 mm/s. Analysis of plaque morphology was performed as previously described (Tearney et al., 2012; Milzi et al., 2017). In particular, as widely employed in clinical practice and in previous intravascular imaging studies, FCT is measured at different sites (conventionally 3) per frame, with an analysis performed on different frames in 0.1–0.2 mm intervals. Usually, the rupture is localized in a single point or in a localized area of the cap, not impeding measurement even in the frame(s) with evident rupture, though in slightly different sites. In order to combine different features of plaque vulnerability for comparison with results of morpho-mechanical analysis, we calculated two different, established scores. The score validated in a publication from Burgmaier et al. (following: Burgmaier Score) is calculated as −2.401 + 1.568 * (insert one if macrophages present; else 0) + 2.639 * (medium lipid arc in multiples of 90°) + 0.255 * (lipid plaque length in mm) − 0.738 * (minimal FCT in multiples of 10 μm), as previously described (Burgmaier et al., 2014). The score based on the CLIMA study (following: CLIMA score) attributed one point each to the presence of MLA < 3.5 mm2, FCT < 75 µm, lipid arc circumferential extension > 180°, and presence of OCT-defined macrophages (Prati et al., 2020). Manual segmentation and reconstruction Request a detailed protocol In order to obtain a patient-specific model of the coronary plaque, we selected a single frame from every OCT pullback. In ruptured plaques, the frame immediately preceding the site of the rupture was chosen; this was based on the consideration that, due to rupture of the fibrous cap and to the present artifacts (caused for instance by thrombotic material), exact reconstruction of the rupture site may be inaccurate. For stable lesions, the frame with the minimal lumen area was selected; this was based on the need to select in a uniform way the site with the most advanced plaque development, assuming only a negative remodeling. After image selection, the operator (AM) was blinded to clinical presentation, as each frame was marked with a random ID. The selected image was then scaled 10:1 previous to segmentation of the different components of the atherosclerotic plaque, which was manually performed using commercial software (AutoCAD 2017, AutoDesk INC, San Rafael, CA). Plaque composition was analyzed
Two-photon (2P) lithography shows great potential for the fabrication of three-dimensional (3-D) micro- and nanomechanical elements, for applications ranging from microelectromechanical systems to tissue engineering, by virtue of its high resolution (<;100 nm) and biocompatibility of the photosensitive resists. However, there is a considerable lack of quantitative data on mechanical properties of materials for 2P lithography and of structures obtained through this technique. In this paper, we combined static and dynamic mechanical analysis on purpose-designed microstructures (microbending of pillar-like structures and picometer-sensitive laser Doppler vibrometry of drum-like structures) to viably and nondestructively estimate Young's modulus, Poisson's ratio, and density of materials for 2P lithography. This allowed us to analyze several polymeric photoresists, including acrylates and epoxy-based materials. The experiments reveal that the 2P exposure power is a key parameter to define the stiffness of the realized structures, with hyperelasticity clearly observable for high-power polymerization. In the linear elastic regime, some of the investigated materials are characterized by a quasi-linear dependence of Young's modulus on the used exposure power, a yet unknown behavior that adds a new degree of freedom to engineer complex 3-D micro- and nanomechanical elements.
