{"paper_id":"2f64262a-ee03-4afb-b20d-9ac1fda67e16","body_text":"1 \n \nStress-free state in human carotid arteries cannot be revealed \nwithout layer separation \n \nAnna Hrubanová1*, Ondř ej Lisický1, Ondř ej Sochor1, Zdeně k Bednař ík2, Marek Joukal3, Jiř í \nBurša1 \n1 Institute of Solid Mechanics, Mechatronics and Biomechanics, Faculty of Mechanical \nEngineering, Brno University of Technology, Brno, Czech Republic \n2 First Department of Pathology, St. Anne’s University Hospital, Brno, Czech Republic \n3 Department of Anatomy, Masaryk University, Brno, Czech Republic \n \n*Corresponding author  \nEmail: anna.hrubanova@vutbr.cz \nTel: +420606269960 \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n2 \n \nAbstract 1 \nResidual stresses are considered as a significant factor influencing the stress-states in arteries. 2 \nThese stresses are typically observed through opening angle of a radially cut artery segment, 3 \noften regarded as a primary descriptor of their stress-free state. However, the experimental 4 \nevidence regarding the stress-free states of different artery layers is scarce. In this study, two 5 \nexperimental protocols, each employing different layer-separating sequences, were performed 6 \non 17 human common carotid arteries; the differences between both protocols were found 7 \nstatistically insignificant. While the media exhibited opening behaviour (reduced curvature), a 8 \ncontrasting trend was observed for the adventitia curvature, indicating its closing behaviour. 9 \nIn addition to the different bending effect, length changes of both layers after separation were 10 \nobserved, namely shortening of the adventitia and elongation of the media. The results point 11 \nout that not all the residual stresses are released after a radial cut but a significant portion of 12 \nthem is released only after the layer separation.  Considering the different mechanical 13 \nproperties of layers, this may significantly change the stress distribution in arterial wall and 14 \nshould be considered in its biomechanical models. 15 \nKeywords 16 \nCommon carotid artery, layer specific residual deformation, vascular heterogeneity  17 \n1. Introduction 18 \nBiomechanical investigation of the presence of residual stresses (RSs) in arteries dates back 19 \nseveral decades (1–3). Arterial vessels, whether in situ or post-extraction, cannot be deemed 20 \nstress-free due to the existence of  RSs in the vessel walls (4,5); they are manifested by the 21 \ntypical opening of a circumferential strip of the artery wall documented in various 22 \nexperimental studies (4,6). Although absolute values of RSs may be smaller in comparison to 23 \nthe stresses experienced by arterial wall in vivo , their significance in shaping the resulting 24 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n3 \n \nstress distribution should not be overlooked. The strain stiffening response of arterial tissue 25 \namplifies the impact of RSs, rendering the stress distribution more uniform throughout the 26 \nwall  thickness (5–8). 27 \nPatient-specific computational models of pathological arteries have confirmed their capability 28 \nto predict arterial tissue rupture. For instance, Polzer et al. (9) demonstrated better rupture risk 29 \nprediction of abdominal aortic aneurysm (AAA) using a novel probabilistic rupture risk 30 \nindex, outperforming the current treatment guidelines (10). A similar predictive approach 31 \ncould be applied to assess the vulnerability of atheromas, particularly those located in life-32 \nthreatening locations such as coronary or carotid arteries. The inclusion of RSs in 33 \ncomputational modelling is necessary, as the initial stress-free state is a prerequisite for any 34 \ncomputational model (11). However, multiple studies seem to overlook the effect of RSs and 35 \nstrains in atherosclerotic carotid arteries (12–17) when evaluating their mechanical response 36 \nto in-vivo mechanical loading.  Although efforts have been made to incorporate the RSs into 37 \nfinite element (FE) models (11,18–22), proving the importance of RS in the resulting stress 38 \ndistribution, the scarcity of experimental data demonstrating RSs in the simulated artery types 39 \nremains a challenge.  40 \nMost experiments focus on the entire artery wall without specific layer separation, thereby 41 \nneglecting the multi-layered heterogeneous structure of the arterial wall. An experimental and 42 \ncomputational study involving the complete human and porcine carotid artery bifurcation (5) 43 \naffirmed that the inclusion of RSs in the computational models leads to a more uniform stress 44 \ndistribution and reduces maximal stress values on the inner wall surface. Greenwald et al. (4) 45 \nexplored different opening angles in two layers of bovine carotid arteries, noting larger 46 \nopening angles in the inner layers compared to the outer ones. Holzapfel et al. (6) 47 \nexperimentally examined layer-specific residual deformations in human aortas, specifically 48 \nexploring the opening angles of axial and circumferential strips from intima, media and 49 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n4 \n \nadventitia layers. The methodology was further refined in (23) and applied to human carotid 50 \narteries in (24), where the adventitia (A) and media + intima (MI) layers were investigated 51 \nseparately. They recorded significant curvature changes after having released residual 52 \nstretches in both axial and circumferential directions for both A and MI layers. 53 \nHowever, a deeper experimental investigation of this phenomenon is essential to gather data 54 \napplicable as input for simulations of stresses in arteries with atheroma, as done, for instance, 55 \nby Pierce et al. (19) and Sigaeva et al. (20). Approaches based on stress homogenization 56 \nthroughout the wall thickness, as seen in (25), may falter in highly non-homogeneous arteries 57 \nwith atheromas. The review (26) focusing on atherosclerosis does mention RS yet exclusively 58 \nfor the non-atherosclerotic tissue.  59 \nHence, the objective of this study is to compare different experimental procedures ensuring 60 \ncredible experimental data on RSs demonstrated by residual deformation in separate layers, 61 \nspecifically in the circumferential direction, for healthy carotid arteries. Such insights can 62 \nprove invaluable for computational modelling of arteries afflicted with atheroma and 63 \npredictions of atheroma vulnerability.  64 \n2. Materials and Methods  65 \nFor the purpose of this study, 17 common carotid arteries (CCAs) were harvested (between 66 \n1.4.2021 and 31.3.2024) during autopsies at Masaryk University, Department of Anatomy, 67 \nwith the approval of the local Ethics committee. Informed written consent from the body 68 \ndonors was obtained in Anatomy Bequest Program years prior to their death.  The donor 69 \ncohort comprised 8 males and 7 females, with an average age of 80.7±7.5 years. The samples 70 \nwere preserved frozen in a saline solution (0.9% NaCl) at -20°C since it was not possible to 71 \ntest the samples immediately after extraction. Some studies suggest that freezing may change 72 \nthe mechanical properties (27–30), however the change was observed in longitudinal direction 73 \nonly or at higher strains (28,29), or was mostly statistically insignificant (30). Other studies 74 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n5 \n \nlean towards the conclusion that freezing at -20°C does not alter mechanical properties (31–75 \n34) and is actually better compared to mere refrigeration (30–32). On the testing day, they 76 \nunderwent a gradual thawing process and were stabilized in the saline solution, maintaining 77 \nthe temperature of 36.7±0.5°C. The samples did not outreach type IV lesion, as per the lesion 78 \ncharacterization according to (35), showing none or mild signs of atherosclerotic changes.  79 \n2.1. Sample preparation and experimental protocol 80 \nThe loose connective tissue was meticulously removed from the outer adventitia surface. Due 81 \nto the limited sample length, varying between 10 and 30 mm, we were unable to obtain both 82 \ncircumferential and axial strips; therefore, only circumferential rings were extracted. Two 83 \ndistinct experimental protocols were implemented (see Fig. 1).  84 \nThe experimental protocol 1 (replicated from (6)) started with cutting the “intact” artery 85 \nsamples into 2 mm height circumferential rings, followed by equilibrating them in a 37°C 86 \nsaline solution for 30 minutes. Subsequently, they were glued pointwise to a plastic cylinder 87 \nusing cyanoacrylate adhesive, followed by a radial incision. This incision induced gradual 88 \nopening of the rings, manifesting thus a release of circumferential RS, tensile at the outer and 89 \ncompressive at the inner artery surface. After 16 hours, the opened configuration was 90 \ndocumented, and the adventitia was separated from the MI layer. Another recording occurred 91 \nafter additional 6 hours in the tempered bath.  92 \nIn the experimental protocol 2, the A and MI layers were separated first. Attempts to isolate 93 \nthe intima from the media without damage were hindered by the natural thinness of the 94 \ncarotid intima. Moreover, the initial atherosclerotic stages related to patients’ age also blur the 95 \nintima-media interface. Similarly to other studies (24), the inner layer was denoted as the MI 96 \nlayer. Then 2 mm circumferential strips were cut from both A and MI layers and glued 97 \npointwise to a cylindrical plastic tube. Images [dataset ] were captured using a CCD camera 98 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n6 \n \n(resolution 1280x1024) perpendicular to the tested ring 30 minutes, 16 hours and 22 hours 99 \nafter the radial incision, aligning with the intervals applied in (6) and maintaining time 100 \nconsistency across both protocols. 101 \nThe thickness of the unseparated ring and of both layers was measured by a dial indicator at 102 \nthree locations for each specimen, the average value was considered for the subsequent 103 \nanalysis. The layer separation was examined by histological analysis (Fig. 2C, D) (standard 104 \nhaematoxylin and eosin stain) and quantified by the thickness ratio of the separated MI and A 105 \nlayers. A total of 22 intact specimens were prepared for protocol 1, yielding 21 adventitia and 106 \n20 media specimens; for the second protocol, 27 media samples and 25 adventitia samples 107 \nwere prepared. The specimen discard was due to occasional tissue damage and unsuccessful 108 \nlayer separation. The adventitia layer, distinguishable from the media by colour and texture, 109 \ncan be easily separated in a “turtleneck fashion” (24) (Fig. 2A). The media layer is stiffer and 110 \nmaintains its cylindrical shape even post-separation, whereas the adventitia becomes flat 111 \nunder gravity  112 \n(Fig. 2B). It was observed that upon repeated immersion in the saline solution, the adventitia 113 \nlayer regains its cylindrical shape, demonstrating its high compliance.  114 \nFig. 1: Two distinct experimental protocols were employed in this study to investigate the 115 \nresidual deformations of carotid wall layers and to compare different RS releasing 116 \nprocedures. The A (blue) and MI (yellow) layers were recorded within 22 hours after being 117 \nradially cut. Black circles represent plastic cylinders used for specimen fixation. 118 \nGiven the temperature-dependent nature of arterial mechanical responses (36,37), specimens 119 \nwere kept in the 37°C saline solution throughout testing for both experimental protocols, 120 \npreventing tissue dehydration.  121 \nFig. 2: The adventitia and media separation in a \"turtleneck fashion” (A). The adventitia 122 \nlayer turned inside-out for removal of the remaining media (B).  Histological examination of 123 \nadventitia (C) and whole artery wall (D) showed some remaining portions of the media on the 124 \nadventitia. 125 \n 126 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n7 \n \n2.2.  Image processing  127 \nEach acquired specimen image underwent analysis using an in house semi-automatic program 128 \nin Python to obtain essential geometric parameters. The opening angle Φ  [°] was defined by 129 \ntwo lines pointed from the middle of the inner boundary to both ending points (equidistant). 130 \nThe definitions of the opening angle vary among studies, but they can be recalculated (8) for 131 \ncomparison, assuming the circular shape. However, local curvature represents a better RS 132 \nquantifier independent of the strip length and asymmetry of the specimens (24); this is 133 \nimportant especially for non-homogeneous atherosclerotic CCA walls.  For this purpose, the 134 \ninner and outer artery borders were manually traced, ensuring enough points (approx. 20 for 135 \neach) for their accurate description. Subsequently, B-splines were utilized to approximate 136 \nthese traced borders and divided by 40 equidistant points. The sum of lengths between these 137 \npoints was considered as the approximate specimen length. Image scale was obtained using 138 \nthe known diameter of the plastic cylindric tube. Local curvature [mm -1] was calculated at 139 \neach of the points using equation (1):  140 \n /g3404\n/g3051 /g4594/g3052 /g4594/g4594/g2879/g3052 /g4594/g3051 /g4594/g4594\n/g4672/g3051 /g4594/g3118/g2878/g3052 /g4594/g3118/g4673\n/g3119\n/g3118\n           ( 1 )  141 \nwhere /g1876 /g4593 , /g1877 /g4593  and /g1876 /g4593/g4593 , /g1877 /g4593/g4593  are the first and second derivatives of the B-spline, respectively. 142 \nSubsequently, a local radius of curvature [mm] was calculated as follows:  143 \n/g1844/g3404\n/g2869\n             ( 2 )  144 \nwhich was then averaged across all the calculated points within each segment boundary. 145 \nTherefore, we evaluated local curvatures of the B-spline of the respective line (38 points for 146 \neach B-spline); the averaged value represents then a global characteristic.  147 \nIn summary, the six parameters of interest for both detached layers included their thickness, 148 \ninner and outer curvature, inner and outer length, and opening angle.  149 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n8 \n \n2.3. Statistical analysis 150 \nStatistical analyses were conducted using the Minitab software. The Anderson-Darling 151 \nnormality test was employed for each dataset to ascertain a normal distribution of the data. If 152 \nthe normal distribution was confirmed, the data was presented as mean ± standard deviation; 153 \notherwise, the median and interquartile range were used. To assess differences in thickness, 154 \ncurvature and the opening angle of layers between both experimental protocols, a 2-sample t-155 \ntest was applied when normal distribution was confirmed; otherwise, the Mann-Whitney test 156 \nwas employed. For variables measured within the same sample (e.g. length before and after 157 \nthe release of RS), the paired t-test was used for normally distributed data, and the Wilcoxon 158 \nsigned-rank test served as the nonparametric alternative.  159 \nFurthermore, Spearman's correlation coefficient ρ  was used for identification of a potential 160 \nrelationship between the opening angle and sample thickness. In all cases, statistical 161 \nsignificance was considered if p < 0.05. 162 \n3. Results  163 \nThickness 164 \nHistological examination indicated that achieving a perfect separation of the adventitia layer 165 \nwas challenging as some portions of the media consistently adhered to the adventitia layer  166 \n(Fig. 2C). Despite meticulous efforts, achieving a flawless separation was deemed 167 \nunattainable even under the supervision of an experienced surgeon from St. Anne’s 168 \nUniversity Hospital. As the media consists of multiple very thin elastin membranes, they 169 \nenable different separation planes and are prone to tearing (Fig. 2D). Despite the efforts to 170 \nclean the media from the adventitia post-separation, the thickness variations of the adventitia 171 \nlayer persisted across specimens pointing out the inability of cleaning the retained media 172 \ncompletely. A 2-sample t-test indicated no significant difference in thickness of the specific 173 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n9 \n \nlayers (MI and A) between both protocols 1 and 2. The mean thickness values for MI, A and 174 \nthe intact artery wall (I) were 0.60±0.14 mm, 0.36±0.11 mm and 0.92±0.15 mm, respectively. 175 \nOpening angle  176 \nThe opening angles for both experimental protocols are shown in Fig. 3, displaying values for 177 \ndetached layers as well as intact artery walls (for protocol 1). Predominantly positive values 178 \nwere observed for the media and even slightly higher for the intact wall. Conversely, negative 179 \nvalues predominated for the adventitia, indicating strip closure rather than opening (Fig. 7C or 180 \nH). Absolute values of both opening and closing angles increased in time for both layers and 181 \nexperimental protocols, which were compared for the separate layers in the final states. For 182 \nboth layers, the statistical analysis revealed no significant difference between the two 183 \nprotocols, therefore they were merged; the median of MI opening angle was 42.17° (13.38, 184 \n97.27), and the mean opening angle of A was -5.19±30.11°. For the intact wall, i.e. protocol 1 185 \nafter 16 hours, the opening angle was 53.05±35.84°. 186 \nFig. 3: Opening angle [°] for both MI and A layers as well as intact ring I (in experimental 187 \nprotocol 1). 188 \nCurvature  189 \nCurvature of the inner boundary is summarized in Fig. 4. The trends are opposite to those of 190 \nthe opening angles because of their inverse relation. Similarly to the opening angles, 191 \ncomparison of the final values (after 22 hours) revealed no statistically significant differences 192 \nfor both MI and A layers between the two experimental protocols, thus their values were 193 \nmerged. The curvature was 0.243±0.105 mm -1 for the MI layer, 0.343±0.071 mm -1 for the A 194 \nlayer, and 0.237±0.080 mm -1 for the intact wall (protocol 1 only). Similar results were 195 \nobserved for the outer boundary with curvature values of 0.237±0.145 mm -1, 0.280±0.058 196 \nmm-1 and 0.181±0.056 mm-1 for the MI and A layers and the intact artery wall, respectively. 197 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n10 \n \nFig. 4: Curvature [mm -1] for both experimental protocols (protocol 1 is on grey 198 \nbackground) – inner boundary (top), outer boundary (bottom). 199 \nImpact of imperfect separation  200 \nVisual inspection, histology analysis and thickness measurement revealed varying amounts of 201 \nmedia remaining on the adventitia layer (see Fig. 2C). The MI/A thickness ratio 202 \napproximately quantifies this portion, with lower values indicating the adventitia layer more 203 \npolluted with residual media. A positive correlation (ρ  = 0.625; p < 0.005) was found between 204 \nthe adventitia curvature and the thickness ratio (see Fig. 5), suggesting that lower curvatures 205 \n(i.e. positive opening angles) of adventitia are linked to a higher portion of residual media. 206 \nConversely, for a lower amount of residual media, opening angles were mostly negative 207 \naccording to their negative correlation with the thickness ratio (ρ  = -0.432, p < 0.005). For the 208 \nMI layer, trends were opposite to those found for adventitia, negative for the curvature  209 \n(ρ  = -0.419, p = 0.011) and positive for the opening angle. However, this last correlation was 210 \nnot statistically significant ( ρ  = 0.273, p = 0.069), although the p-value was close to the 211 \nsignificance level of 0.05. 212 \nFig. 5: Correlation between thickness ratio and the opening angle (left) and inner curvature 213 \n(right). The higher the thickness ratio, the lower portion of media remains on the adventitia 214 \nlayer. 215 \nLength of specimens 216 \nTime development of the length of specimens in the circumferential direction at their inner 217 \nand outer boundaries is presented in Fig. 6. Comparisons of inner and outer boundary lengths 218 \ndid not reveal significant differences between experimental protocols 1 and 2 thus, analogous 219 \nto curvature and opening angle, the data was merged. The mean length of the inner boundary 220 \nin the final stage was 18.404±2.703 mm for media, 19.654±2.280 mm for adventitia and 221 \n18.471±2.754 mm for the intact wall (protocol 1 only). For the outer boundary, the mean 222 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n11 \n \nvalues were 21.845±2.387 mm, 24.205±2.983 mm and 23.869±2.695 mm for media, 223 \nadventitia, and intact wall, respectively.  224 \nFig. 6: Length [mm] of the specimens for both experimental protocols (protocol 1 is on grey 225 \nbackground) – inner boundary (top), outer boundary (bottom). 226 \n 227 \nIn addition, comparison between the length of the initial uncut ring and the length of the final 228 \nstate (after RS release) was conducted for each specimen using a paired t-test (for MI and A 229 \nlayers in protocol 2 and for I in protocol 1). The test revealed significant differences in 230 \nlengths for all cases (MI, A, and I) at the inner boundary, while at the outer boundary, only A 231 \nand I exhibited statistically significant differences. The inner boundary of the MI layer 232 \nelongated by 6.12%, while the outer boundary remained unchanged. For the A layer, both the 233 \ninner and outer boundaries shortened by 3.80% and 3.81%, respectively. The inner boundary 234 \nof I elongated by 6.18%, whereas the outer boundary shortened by 4.18%. While the strain 235 \ndistribution in the media showed mostly a bending nature, in the adventitia, both bending and 236 \nstretching were evident. 237 \n4. Discussion 238 \nKnowledge on the mechanical behaviour of arterial wall is essential for its computational 239 \nmodelling, where RSs are generally accepted to play a significant role, and their inclusion in 240 \ncomputational models is often recommended. However, experimental evidence, especially in 241 \nspecific carotid artery layers and atheromatous arteries, remains sparsely explored and 242 \ncontradictory. Existing studies are based either on endarterectomy carotid samples (38), 243 \nwhich exhibit small opening angles, or on coronary arteries (21), characterized by large 244 \nopening angles. However, specimens from endarterectomy show much higher degree of 245 \natherosclerosis and incorporate atheroma instead of adventitia (and partially media), thus the 246 \nresults in (39) correspond rather to our MI layer. The same models and approaches were used 247 \nfor mouse samples in (40), recommending (together with (41)) the use of circumferential RS 248 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n12 \n \nwhen analysing healthy and diseased arterial tissue. However, images of a closed segment 249 \npublished in (41) show some buckling effects causing unrealistic penetration of the fibrous 250 \ncap (FC) or even lipid core into the lumen area. These effects were not discussed, although 251 \nthey indicate large negative stresses in the FC instead of positive stresses expected at FC 252 \nrupture. This contradiction requires further experimental and computational investigation of 253 \nthis phenomenon. 254 \nIn this study, two experimental protocols evaluating RS were compared. The first 255 \nexperimental protocol, echoing the methodology employed in (6), involves layer separation 256 \n16 hours after the radial cut, as opposed to the second experimental protocol introduced in this 257 \nstudy, which opts for cutting the rings after the separation of layers. When the radial cut is 258 \nmade first and the separation of layers follows, the initial RS release happens while the layers 259 \nare still joined together and influence each other. However, no statistically significant 260 \ndifference was found between final stages of both experimental protocols (after 22 hours) for 261 \nany of the investigated parameters (thickness; opening angle; inner and outer curvature; inner 262 \nand outer length) for both A and MI layer. Differences are in the attainable results: while the 263 \nfirst method mediates insights into the intact wall, the second unfolds layer-specific 264 \ndeformation, which is of dominantly bending character in the intact wall as well as in the MI 265 \nlayer. However, the A shows much lower bending stiffness (it is proportional to the third 266 \npower of the layer thickness) and much lower average opening angle values (or change of 267 \ncurvature). , Consequently, the adventitia deformation is dominated by contraction with a 268 \nnearly constant average value of 3.8 % throughout the thickness and considering this effect on 269 \nthe RS distribution may be more important than the opening angle (or change of curvature) of 270 \nthe A layer.  271 \nWhen comparing adventitia and media, both layers show statistically significant differences 272 \nfor all the six parameters. The thickness was found to be 0.60±0.14 mm for the MI and 273 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n13 \n \n0.36±0.11 mm for the adventitia. These results corroborate that adventitia is much thinner 274 \nthan the MI layer. Nevertheless, our histology analysis revealed that the adventitia separation 275 \nwas not perfect (Fig. 2C) although the thicknesses are comparable with the values (0.7±0.13 276 \nmm and 0.47±0.07 mm, respectively) published in (24). As our MI/A thickness ratio was even 277 \nhigher than in that study, it suggests neither their separation was perfect. 278 \nThe tendency of adventitia to negative angles (closing rather than opening, see Fig. 7 C, H) is 279 \nnot yet thoroughly described in literature. For instance, Teng et al. (39), although focused 280 \nmainly on mechanical characterization (strength and stress-strain response) of both layers, 281 \nmentioned the opening effect for the whole wall, as well as for the separated layers. However, 282 \ntheir provided photo documentation is ambiguous, one figure shows an opened adventitia 283 \nsegment while another shows its zero opening. As they reported equal thickness of MI and A, 284 \ntheir results correspond to ours with the lowest MI/A thickness ratio of approximately 1 (i.e. a 285 \nsignificant portion of residual media on the adventitia), which gave also nearly zero angles in 286 \nour study. It is also unclear whether their figures were taken in the solution or in the air, thus 287 \npossibly influencing the results.  Kural et al. (42)  focused mainly on biaxial tensile tests but 288 \nbriefly mentioned RS characterized by the opening angle. A positive mean opening angle of 289 \n63° was reported for intact carotid artery walls while for separated layers some retraction or 290 \nexpansion was mentioned without being specified or further discussed. Esmaeili Monir et al. 291 \n(18) focused on finite element modelling but showed one CCA sample with separated 292 \nadventitia, media and intima. Here, a negative opening angle of -13° was recorded for 293 \nadventitia which confirms our results. 294 \nFig. 7: Example of typical opening of segments glued pointwise at the outer surface to a 295 \nplastic cylinder.  A, B: Intact wall – ring and after 16 hours; C, D: Adventitia and MI – 6 296 \nhours after separation, respectively; E, F: MI – ring and after 22 hours; G, H: Adventitia – 297 \nring and after 22 hours. 298 \n 299 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n14 \n \nSommer et al. (24) presented a decrease of curvature (opening tendency) in both MI and A 300 \nlayers of CCA immediately after their separation while some tendency to its re-increase with 301 \ntime occurred. They stated a slight increase of adventitia curvature (mean values) within 6 302 \nhours after separation, supporting thus partially the results of our study, in which we also 303 \naccepted their approach applying the average curvature as RS quantifier. Although the final 304 \ncurvature was slightly higher than that of the intact wall, it was still much lower than the 305 \ncurvature of the uncut ring and represented thus an opening behaviour of adventitia. However, 306 \nthe specimens were glued on the inner surface to a plastic cylinder with diameter comparable 307 \nto their inner diameter, thus a larger closing of the ring (negative opening angle) was 308 \nprecluded, and no statistical analysis was presented either. Statistical test of our results 309 \ncomparing the curvature of the inner adventitia boundary (in the stable state of experimental 310 \nprotocol 2) with the uncut adventitia ring confirmed the closing behaviour of adventitia 311 \nindicating a significant release of RS. The same test done for the outer adventitia boundary 312 \nhas not reached statistical significance, because the remaining connective tissue makes the 313 \nboundary blurry. Moreover, it induces some friction and constrains thus mutual movement of 314 \nthe free ends. This behaviour was noted when the free ends were mutually pushed together, 315 \ndistorting thus the circularity of the ring; the friction prevented the segment from further 316 \nclosing even though such tendency was apparent.  317 \nWhen comparing the length of the uncut adventitia ring and the length of the stable state cut 318 \nspecimen, the adventitia strips shortened significantly. This behaviour was also supported by 319 \nthe results from the intact wall specimens; here the statistical test revealed a difference 320 \nbetween the inner and outer boundaries. The inner boundary (intima) elongated by 6.18%, 321 \nwhereas the outer boundary (adventitia) shortened by 4.18%. When comparing also the results 322 \nfrom the separated MI layer, which elongates on the inner boundary but keeps the same length 323 \nof the outer boundary, the following conclusions can be drawn: the intima on the inner surface 324 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n15 \n \ntends to elongate and, conversely, the adventitia layer shows shortening behaviour. The 325 \nexperimental evidence on the length changes is scarce in literature; (18) reported elongation 326 \nby 2.6% for media, 10.5% for adventitia and shortening by 9.3% for the intima layer for one 327 \nCCA specimen. It is in contradiction to our results; however, it seems these values were read 328 \nshortly after the radial cut; thus, they cannot represent the stable-state values reached much 329 \nlater. Additionally, these layer-specific length values were compared with the lengths of 330 \ndifferent intact specimen taken from the same CCA which may cause a deviation in the 331 \nresults. Sommer et al. (24) reported an axial shortening of the adventitia which was also 332 \nobserved in our experiments but could not be investigated and quantified due to small 333 \ndimension of the specimens; in contrast, shortening in the circumferential direction is not 334 \ndiscussed there. Holzapfel et al. (6) found that adventitia shortens significantly in both 335 \ncircumferential and axial directions after the release of RS, and the intima showed elongation, 336 \nwhich corroborates our results although the media was found to shorten in the circumferential 337 \ndirection. Nevertheless, that study dealt with abdominal aortas differing significantly in 338 \ncomposition and portion of the media layer from the muscular CCA, which may explain these 339 \ndifferences.  340 \nThe length modification was examined only as the uncut ring vs. the cut stable state (in 341 \nprotocol 1 for the intact wall and protocol 2 for the A and MI) allowing us a direct 342 \ncomparison of each sample and thus eliminating the interpatient variation. Surprisingly, when 343 \ncomparing the outer length of MI and the inner length of the A layer (for both separated rings 344 \nbefore radial cut), the adventitia is always shorter (p-value for paired t-test <0.05) with values 345 \nof 22.38±2.57 mm and 20.52±3.03 for MI and A, respectively. Although the layer separation 346 \nof MI and A in the “turtleneck” fashion should not influence the bending RS, the 347 \ntensile/compressive RS are influenced. This means the shortening effect of the A layer 348 \nreaching nearly 10% strain may be even more significant.  349 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n16 \n \nThe circumferential shortening of the adventitia documented in our study may also cause the 350 \ncurvatures of the intact specimen to be smaller than those of media, meaning the intact wall 351 \nshows bigger opening angle than any of its components. The shortening tendency of the 352 \nadventitia pulls the ends of the intact specimen further away from each other, resulting in a 353 \nlarger opening compared with the MI layer. This difference contradicts both hypotheses of 354 \nconstant strain and constant stress in the artery wall (throughout its thickness) and may 355 \nchange completely the RS distribution calculated in biomechanical modelling of artery wall. 356 \nThis impact should be verified in a future study.  357 \n5. Limitations 358 \nAcquisition of human tissue is quite challenging, so the sample number is limited. To increase 359 \nthe number of specimens, we were able to obtain multiple specimens from one sample. 360 \nHowever, consideration of these specimens may induce a bias due to the natural inter-patient 361 \nvariability. Therefore, all the statistical analyses were recalculated on smaller data sets 362 \ncontaining only one specimen per patient to avoid this bias, which allowed the use of paired t-363 \ntest for direct specimen to specimen comparison (Wilcoxon signed-rank test as the 364 \nnonparametric alternative). The results corroborate the conclusions obtained from the full 365 \ndatasets; no statistically significant difference was found between two different experimental 366 \nprotocols (p<0.05) for both MI and A layers when comparing all the parameters of interest: 367 \nthickness, opening angle, inner and outer curvature, inner and outer length; the choice of 368 \nexperimental protocol does not have significant effect. Additionally, the length change after 369 \nradial cut was confirmed on the reduced data as well. The comparison of length of inner 370 \nboundary of A with outer boundary length of MI was confirmed on the reduced data set as 371 \nwell; the adventitia was 7.1 % shorter.  372 \nThe study is also limited by using circumferential specimens only, thus further measurements 373 \nof axial residual stresses could enhance the description of the 3D residual stress distribution. 374 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n17 \n \n6. Conclusion 375 \nThe experimental evaluation of the opening angles, curvatures and length changes of 376 \ncircumferential segments of human common carotid arteries and their layers revealed 377 \nsurprisingly increasing curvatures (negative opening angles) for separated adventitia layers. 378 \nThe inner MI layers showed an expectable decrease of curvature (i.e. positive opening 379 \nangles), as well as the segments of intact (unseparated) artery wall, both dominated evidently 380 \nby their bending. Together with significant shortening of adventitia after layer separation, 381 \nthese effects may completely change the distribution of stresses throughout the wall thickness. 382 \nNeither of the hypotheses of constant strain or stress throughout the wall thickness, nor the 383 \nresidual stresses calculated based on opening angle of the artery wall segment are capable to 384 \ndescribe the distribution of residual stresses correctly.  385 \nAcknowledgements 386 \nThis publication was supported by the Czech Science Foundation, research project No. 21-387 \n21935S, as well as by the project \"Mechanical Engineering of Biological and Bio-inspired 388 \nSystems\", funded as project No. CZ.02.01.01/00/22_008/0004634 by Programme Johannes 389 \nAmos Commenius, call Excellent Research and Brno Ph.D. Talent Scholarship – Funded by 390 \nthe Brno City Municipality. 391 \nReferences  392 \n1. Fung YC. On the Foundations of Biomechanics. Journal of Applied Mechanics. 1983 Dec 393 \n1;50(4b):1003–9.  394 \n2. Fung Y. Biodynamics: Circulation. New York: Springer-Verlag; 1984. (Biomechanics).  395 \n3. Chuong CJ, Fung YC. On residual stresses in arteries. J Biomech Eng. 1986 396 \nMay;108(2):189–92.  397 \n4. Greenwald SE, Moore JEJ, Rachev A, Kane TP, Meister JJ. Experimental investigation of 398 \nthe distribution of residual strains in the artery wall. J Biomech Eng. 1997 399 \nNov;119(4):438–44.  400 \n5. Delfino A, Stergiopulos N, Moore JE, Meister JJ. Residual strain effects on the stress 401 \nfield in a thick wall finite element model of the human carotid bifurcation. Journal of 402 \nBiomechanics. 1997;30(8):777–86.  403 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n18 \n \n6. Holzapfel GA, Sommer G, Auer M, Regitnig P, Ogden RW. Layer-specific 3D residual 404 \ndeformations of human aortas with non-atherosclerotic intimal thickening. Ann Biomed 405 \nEng. 2007 Apr;35(4):530–45.  406 \n7. Gasser TC, Ogden RW, Holzapfel GA. Hyperelastic modelling of arterial layers with 407 \ndistributed collagen fibre orientations. Journal of the Royal Society Interface. 2006 Feb 408 \n22;3(6):15–35.  409 \n8. Humphrey JD. Cardiovascular solid mechanics: Cells, tissues, and organs. 1st ed. New 410 \nYork: Springer; 2002. 758 p. Available from: https://doi.org/10.1007/978-0-387-21576-1 411 \n9. Polzer S, Gasser TC, Vlachovský R, Kubí č ek L, Lambert L, Man V, et al. Biomechanical 412 \nindices are more sensitive than diameter in predicting rupture of asymptomatic abdominal 413 \naortic aneurysms. J Vasc Surg. 2020 Feb;71(2):617-626.e6.  414 \n10. Moll FL, Powell JT, Fraedrich G, Verzini F, Haulon S, Waltham M, et al. Management of 415 \nabdominal aortic aneurysms clinical practice guidelines of the European society for 416 \nvascular surgery. Eur J Vasc Endovasc Surg. 2011 Jan;41 Suppl 1:S1–58.  417 \n11. Cilla M, Peña E, Martínez MA. 3D computational parametric analysis of eccentric 418 \natheroma plaque: Influence of axial and circumferential residual stresses. Biomechanics 419 \nand Modeling in Mechanobiology. 2012 Sep;11(7):1001–13.  420 \n12. Sadat U, Li ZY, Young VE, Graves MJ, Boyle JR, Warburton EA, et al. Finite element 421 \nanalysis of vulnerable atherosclerotic plaques: A comparison of mechanical stresses 422 \nwithin carotid plaques of acute and recently symptomatic patients with carotid artery 423 \ndisease. Journal of Neurology, Neurosurgery and Psychiatry. 2010;81(3):286–9.  424 \n13. Noble C, Carlson KD, Neumann E, Lewis B, Dragomir-Daescu D, Lerman A, et al. Finite 425 \nelement analysis in clinical patients with atherosclerosis. J Mech Behav Biomed Mater. 426 \n2022 Jan;125:104927.  427 \n14. Li ZY, Howarth S, Trivedi RA, U-King-Im JM, Graves MJ, Brown A, et al. Stress 428 \nanalysis of carotid plaque rupture based on in vivo high resolution MRI. J Biomech. 429 \n2006;39(14):2611–22.  430 \n15. Kock SA, Nygaard JV, Eldrup N, Fründ ET, Klaerke A, Paaske WP, et al. Mechanical 431 \nstresses in carotid plaques using MRI-based fluid-structure interaction models. J 432 \nBiomech. 2008;41(8):1651–8.  433 \n16. Kiousis DE, Rubinigg SF, Auer M, Holzapfel GA. A methodology to analyze changes in 434 \nlipid core and calcification onto fibrous cap vulnerability: the human atherosclerotic 435 \ncarotid bifurcation as an illustratory  example. J Biomech Eng. 2009 Dec;131(12):121002.  436 \n17. Auricchio F, Conti M, De Beule M, De Santis G, Verhegghe B. Carotid artery stenting 437 \nsimulation: From patient-specific images to finite element analysis. Medical Engineering 438 \nand Physics. 2011;33(3):281–9.  439 \n18. Esmaeili Monir H, Yamada H, Sakata N. Finite element modelling of the common carotid 440 \nartery in the elderly with physiological intimal thickening using layer-specific stress-441 \nreleased geometries and nonlinear elastic properties. Computer Methods in Biomechanics 442 \nand Biomedical Engineering. 2016 Sep 9;19(12):1286–96.  443 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n19 \n \n19. Pierce DM, Fastl TE, Rodriguez-Vila B, Verbrugghe P, Fourneau I, Maleux G, et al. A 444 \nmethod for incorporating three-dimensional residual stretches/stresses into patient-445 \nspecific finite element simulations of arteries. Journal of the Mechanical Behavior of 446 \nBiomedical Materials. 2015 Jul 1;47:147–64.  447 \n20. Sigaeva T, Sommer G, Holzapfel GA, Di Martino ES. Anisotropic residual stresses in 448 \narteries. Journal of the Royal Society Interface. 2019 Feb 1;16(151).  449 \n21. Ohayon J, Dubreuil O, Tracqui P, Le Floc’ S, Rioufol G, Chalabreysse L, et al. Influence 450 \nof residual stress/strain on the biomechanical stability of vulnerable coronary plaques: 451 \npotential impact for evaluating the risk of plaque rupture. Am J Physiol Heart Circ 452 \nPhysiol. 2007;293:1987–96.  453 \n22. Patel SY, Kaazempur-Mofrad MR, Isasi AG, Kamm RD. DISEASED ARTERY WALL 454 \nMECHANICS: CORRELATION TO HISTOLOGY. In: Book of abstracts. Key 455 \nBiscayne, Florida; June 25-29. (CARDIOVASCULAR SOLID/FLUID 456 \nINTERACTIONS).  457 \n23. Holzapfel GA, Ogden RW. Modelling the layer-specific three-dimensional residual 458 \nstresses in arteries, with an application to the human aorta. J R Soc Interface. 2010 May 459 \n6;7(46):787–99.  460 \n24. Sommer G, Regitnig P, Költringer L, Holzapfel GA. Biaxial mechanical properties of 461 \nintact and layer-dissected human carotid arteries at physiological and supraphysiological 462 \nloadings. American Journal of Physiology - Heart and Circulatory Physiology. 463 \n2010;298(3):898–912.  464 \n25. Schröder J, von Hoegen M. An engineering tool to estimate eigenstresses in three-465 \ndimensional patient-specific arteries. Computer Methods in Applied Mechanics and 466 \nEngineering. 2016 Jul 1;306:364–81.  467 \n26. Holzapfel GA, Mulvihill JJ, Cunnane EM, Walsh MT. Computational approaches for 468 \nanalyzing the mechanics of atherosclerotic plaques: A review. Journal of Biomechanics. 469 \n2014;47(4):859–69.  470 \n27. Venkatasubramanian RT, Grassl ED, Barocas VH, Lafontaine D, Bischof JC. Effects of 471 \nfreezing and cryopreservation on the mechanical properties of arteries. Annals of 472 \nBiomedical Engineering. 2006 May;34(5):823–32.  473 \n28. Chow MJ, Zhang Y. Changes in the mechanical and biochemical properties of aortic 474 \ntissue due to cold storage. Journal of Surgical Research. 2011 Dec;171(2):434–42.  475 \n29. Grassl ED, Barocas VH, Bischof JC. Effects of Freezing on the Mechanical Properties of 476 \nBlood Vessels. In: IMECE2004. Heat Transfer, Volume 1; 2004. p. 699–703. Available 477 \nfrom: https://doi.org/10.1115/IMECE2004-60244 478 \n30. Hemmasizadeh A, Darvish K, Autieri M. Characterization of changes to the mechanical 479 \nproperties of arteries due to cold storage using nanoindentation tests. Annals of 480 \nBiomedical Engineering. 2012 Jul;40(7):1434–42.  481 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n20 \n \n31. Virues Delgadillo JO, Delorme S, El-Ayoubi R, DiRaddo R, Hatzikiriakos SG. Effect of 482 \nfreezing on the passive mechanical properties of arterial samples. Journal of Biomedical 483 \nScience and Engineering. 2010;03(07):645–52.  484 \n32. Stemper BD, Yoganandan N, Pintar FA. Methodology to study intimal failure mechanics 485 \nin human internal carotid arteries. Journal of Biomechanics. 2005;38(12):2491–6.  486 \n33. O’Leary SA, Doyle BJ, McGloughlin TM. The impact of long term freezing on the 487 \nmechanical properties of porcine aortic tissue. Journal of the Mechanical Behavior of 488 \nBiomedical Materials. 2014;37:165–73.  489 \n34. Ebenstein DM, Coughlin D, Chapman J, Li C, Pruitt LA. Nanomechanical properties of 490 \ncalcification, fibrous tissue, and hematoma from atherosclerotic plaques. Journal of 491 \nBiomedical Materials Research - Part A. 2009;91(4):1028–37.  492 \n35. Stary HC. Natural history and histological classification of atherosclerotic lesions: an 493 \nupdate. Arterioscler Thromb Vasc Biol. 2000 May;20(5):1177–8.  494 \n36. Atienza JM, Guinea GV, Rojo FJ, Burgos RJ, García-Montero C, Goicolea FJ, et al. The 495 \ninfluence of pressure and temperature on the behavior of the human aorta and carotid 496 \narteries. Revista Española de Cardiología (English Edition). 2007;60(3):259–67.  497 \n37. Guinea GV, Atienza JM, Elices M, Aragoncillo P, Hayashi K. Thermomechanical 498 \nbehavior of human carotid arteries in the passive state. American Journal of Physiology - 499 \nHeart and Circulatory Physiology. 2005;288(6 57-6):2940–5.  500 \n38. Pocaterra M, Gao H, Das S, Pinelli M, Long Q. Circumferential residual stress 501 \ndistribution and its influence in a diseased carotid artery. 2009. (Summer bioengineering 502 \nconference; vol. ASME 2009 Summer Bioengineering Conference, Parts A and B). 503 \nAvailable from: https://doi.org/10.1115/SBC2009-206692 504 \n39. Teng Z, Tang D, Zheng J, Woodard PK, Hoffman AH. An experimental study on the 505 \nultimate strength of the adventitia and media of human atherosclerotic carotid arteries in 506 \ncircumferential and axial directions. Journal of Biomechanics. 2009;42(15):2535–9.  507 \n40. Broisat A, Toczek J, Mesnier N, Tracqui P, Ghezzi C, Ohayon J, et al. Assessing low 508 \nlevels of mechanical stress in aortic atherosclerotic lesions from apolipoprotein E-/- mice-509 \nbrief report. Arteriosclerosis, Thrombosis, and Vascular Biology. 2011;31(5):1007–10.  510 \n41. Martiel JL, Finet G, Holzapfel GA, Stuber M, Matsumoto T, Pettigrew RI, et al. Chapter 511 \n19 - Importance of residual stress and basal tone in healthy and pathological human 512 \ncoronary arteries. In: Ohayon J, Finet G, Pettigrew RI, editors. Biomechanics of coronary 513 \natherosclerotic plaque. Academic Press; 2021. p. 433–61. (Biomechanics of living organs; 514 \nvol. 4). Available from: 515 \nhttps://www.sciencedirect.com/science/article/pii/B9780128171950000196 516 \n42. Kural MH, Cai M, Tang D, Gwyther T, Zheng J, Billiar KL. Planar biaxial 517 \ncharacterization of diseased human coronary and carotid arteries for computational 518 \nmodeling. Journal of Biomechanics. 2012;45(5):790–8.  519 \n[dataset] A. Hrubanová, O. Sochor, Raw images of CCA specimens, Zenodo, (2024).  520 \n https://doi.org/10.5281/zenodo.10932993. 521 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 17, 2024. ; https://doi.org/10.1101/2024.10.15.618414doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}