The time has come to extend the expiration limit of cryopreserved allograft heart valves.

Despite the wide choice of commercial heart valve prostheses, cryopreserved semilunar allograft heart valves (C-AHV) are required, and successfully transplanted in selected groups of patients. The expiration limit (EL) criteria have not been defined yet. Most Tissue Establishments (TE) use the EL of 5 years. From physiological, functional, and surgical point of view, the morphology and mechanical properties of aortic and pulmonary roots represent basic features limiting the EL of C-AHV. The aim of this work was to review methods of AHV tissue structural analysis and mechanical testing from the perspective of suitability for EL validation studies. Microscopic structure analysis of great arterial wall and semilunar leaflets tissue should clearly demonstrate cells as well as the extracellular matrix components by highly reproducible and specific histological staining procedures. Quantitative morphometry using stereological grids has proved to be effective, as the exact statistics was feasible. From mechanical testing methods, tensile test was the most suitable. Young's moduli of elasticity, ultimate stress and strain were shown to represent most important AHV tissue mechanical characteristics, suitable for exact statistical analysis. C-AHV are prepared by many different protocols, so as each TE has to work out own EL for C-AHV.

Persistent occiput posterior position and stress distribution in levator ani muscle during vaginal delivery computed by a finite element model.

INTRODUCTION AND HYPOTHESIS: Objective of this study was to develop an MRI-based finite element model and simulate a childbirth considering the fetal head position in a persistent occiput posterior position. METHODS: The model involves the pelvis, fetal head and soft tissues including the levator ani and obturator muscles simulated by the hyperelastic nonlinear Ogden material model. The uniaxial test was measured using pig samples of the levator to determine the material constants. Vaginal deliveries considering two positions of the fetal head were simulated: persistent occiput posterior position and uncomplicated occiput anterior position. The von Mises stress distribution was analyzed. RESULTS: The material constants of the hyperelastic Ogden model were measured for the samples of pig levator ani. The mean values of Ogden parameters were calculated as: µ1 = 8.2 ± 8.9 GPa; µ2 = 21.6 ± 17.3 GPa; α1 = 0.1803 ± 0.1299; α2 = 15.112 ± 3.1704. The results show the significant increase of the von Mises stress in the levator muscle for the case of a persistent occiput posterior position. For the optimal head position, the maximum stress was found in the anteromedial levator portion at station +8 (mean: 44.53 MPa). For the persistent occiput posterior position, the maximum was detected in the distal posteromedial levator portion at station +6 (mean: 120.28 MPa). CONCLUSIONS: The fetal head position during vaginal delivery significantly affects the stress distribution in the levator muscle. Considering the persistent occiput posterior position, the stress increases evenly 3.6 times compared with the optimal head position.

Mechanical and structural properties of human aortic and pulmonary allografts do not deteriorate in the first 10 years of cryopreservation and storage in nitrogen.

The aortic and pulmonary allograft heart valves (AHV) are used in the cardiac surgery for replacing the impaired semilunar valves. They are harvested from donor hearts and cryostored in tissue banks. The expiration period was set to 5 years arbitrarily. We hypothesized that their mechanical and structural properties do not deteriorate after this period. A total of 64 human AHV (31 aortic and 33 pulmonary) of different length of cryopreservation (fresh, 0-5, 5-10, over 10 years) were sampled to different tissue strips (artery, leaflet, ventriculo-arterial junction) and tested by tensile test with loading velocity 10 mm/min until tissue rupture. Neighbouring regions of tissue were processed histologically and evaluated for elastin and collagen area fraction. The results were evaluated statistically. In aortic AHV, the physical deformation response of wall samples to stress did not changed significantly neither during the process of cryopreservation nor during the first 10 years of storage. In pulmonary AHV, the ultimate strain dropped after 5 years of cryopreservation indicating that pulmonary artery was significantly less deformable at the time of rupture. On the other hand, the ultimate stress was equal during the first 10 years of cryostorage. The changes in collagen and elastin amount in the tissue samples were not associated with mechanical impairment. Neither elasticity, stiffness and solidity nor morphology of aortic and pulmonary AHV did not change reasonably with cryopreservation and in the first 10 years of cryostorage. This evidence suggests that the expiration period might be extended in the future.

A mathematical model of the carp heart ventricle during the cardiac cycle.

The poikilothermic heart has been suggested as a model for studying some of the mechanisms of early postnatal mammalian heart adaptations. We assessed morphological parameters of the carp heart (Cyprinus carpio L.) with diastolic dimensions: heart radius (5.73mm), thickness of the compact (0.50mm) and spongy myocardium (4.34mm), in two conditions (systole, diastole): volume fraction of the compact myocardium (20.7% systole, 19.6% diastole), spongy myocardium (58.9% systole, 62.8% diastole), trabeculae (37.8% systole, 28.6% diastole), and cavities (41.5% systole, 51.9% diastole) within the ventricle; volume fraction of the trabeculae (64.1% systole, 45.5% diastole) and sinuses (35.9% systole, 54.5% diastole) within the spongy myocardium; ratio between the volume of compact and spongy myocardium (0.35 systole, 0.31 diastole); ratio between compact myocardium and trabeculae (0.55 systole, 0.69 diastole); and surface density of the trabeculae (0.095µm(-1) systole, 0.147µm(-1) diastole). We created a mathematical model of the carp heart based on actual morphometric data to simulate how the compact/spongy myocardium ratio, the permeability of the spongy myocardium, and sinus-trabeculae volume fractions within the spongy myocardium influence stroke volume, stroke work, ejection fraction and p-V diagram. Increasing permeability led to increasing and then decreasing stroke volume and work, and increasing ejection fraction. An increased amount of spongy myocardium led to an increased stroke volume, work, and ejection fraction. Varying sinus-trabeculae volume fractions within the spongy myocardium showed that an increased sinus volume fraction led to an increased stroke volume and work, and a decreased ejection fraction.

Cellular force microscopy for in vivo measurements of plant tissue mechanics.

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM's large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM's ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis.

Thin-layer hydroxyapatite deposition on a nanofiber surface stimulates mesenchymal stem cell proliferation and their differentiation into osteoblasts.

Pulsed laser deposition was proved as a suitable method for hydroxyapatite (HA) coating of coaxial poly-ɛ-caprolactone/polyvinylalcohol (PCL/PVA) nanofibers. The fibrous morphology of PCL/PVA nanofibers was preserved, if the nanofiber scaffold was coated with thin layers of HA (200 nm and 400 nm). Increasing thickness of HA, however, resulted in a gradual loss of fibrous character. In addition, biomechanical properties were improved after HA deposition on PCL/PVA nanofibers as the value of Young's moduli of elasticity significantly increased. Clearly, thin-layer hydroxyapatite deposition on a nanofiber surface stimulated mesenchymal stem cell viability and their differentiation into osteoblasts. The optimal depth of HA was 800 nm.

Porcine spleen as a model organ for blunt injury impact tests: An experimental and histological study.

The spleen is a large and highly vascularized secondary lymphatic organ. Spleen injuries are among the most frequent trauma-related injuries in the abdominal region. The aims of the study were to assess the volume fractions of the main splenic tissue components (red pulp, white pulp, trabeculae and reticular fibres) and to determine the severity of splenic injury due to the experimental impact test. Porcine spleens (n = 17) were compressed by 6.22 kg wooden plate using a drop tower technique from three impact heights (50, 100 and 150 mm corresponding to velocities 0.79, 1.24 and 1.58 m/s). The pressure was measured via catheters placed in the splenic vein. The impact velocity was measured using lasers. The severity of induced injuries was analysed on the macroscopic level. The volume fractions of splenic components were assessed microscopically using stereology. The volume fraction of the red pulp was 76.4%, white pulp 21.3% and trabeculae 2.7% respectively. All impact tests, even with the low impact velocities, led to injuries that occurred mostly in the dorsal extremity of the spleen, and were accompanied by bleeding, capsule rupture and parenchyma crushing. Higher impact height (impact velocity and impact energy) caused more severe injury. Porcine spleen had the same volume fraction of tissue components as human spleen, therefore we concluded that the porcine spleen was a suitable organ model for mechanical experiments. Based on our observations, regions around hilum and the diaphragmatic surface of the dorsal extremity, that contained fissures and notches, were the most prone to injury and required considerable attention during splenic examination after injury. The primary mechanical data are now available for the researchers focused on the splenic trauma modelling.

Decellularization of Porcine Carotid Arteries: From the Vessel to the High-Quality Scaffold in Five Hours.

The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine.

Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

How to asses, visualize and compare the anisotropy of linear structures reconstructed from optical sections--a study based on histopathological quantification of human brain microvessels.

Three-dimensional analyses of the spatial arrangement, spatial orientation and preferential directions of systems of fibers are frequent tasks in many scientific fields, including the textile industry, plant biology and tissue modeling. In biology, systems of oriented and branching lines are often used to represent the three-dimensional directionality and topology of microscopic blood vessels supplying various organs. In our study, we present a novel p(χ²) (chi-square) method for evaluating the anisotropy of line systems that involves comparing the observed length densities of lines with the discrete uniform distribution of an isotropic line system with the χ²-test. Using this method in our open source software, we determined the rose of directions, preferential directions and level of anisotropy of linear systems representing the microscopic blood vessels in samples of various regions from human brains (cortex, subcortical gray matter and white matter). The novel method was compared with two other methods used for anisotropy quantification (ellipsoidal and fractional anisotropy). All three methods detected different levels of anisotropy of blood microvessels in human brain. The microvascular bed in the cortex was closer to an isotropic network, while the microvessels supplying the white matter appeared to be an anisotropic and direction-sensitive system. All three methods were able to determine the differences between various brain regions. The advantage of our p(χ²) method is its high correlation with the number of preferential directions of the line system. However, the software, named esofspy, is able to calculate all three of the measures of anisotropy compared and documented in this paper, thus making the methods freely available to the scientific community.

Blunt injury of liver: mechanical response of porcine liver in experimental impact test.

OBJECTIVE: The liver is frequently injured in blunt abdominal trauma caused by road traffic accidents. The testing of safety performance of vehicles, e.g. belt usage, head support, seat shape, or air bag shape, material, pressure and reaction, could lead to reduction of the injury seriousness. Current trends in safety testing include development of accurate computational human body models (HBMs) based on the anatomical, morphological, and mechanical behavior of tissues under high strain. APPROACH: The aim of this study was to describe the internal pressure changes within porcine liver, the severity of liver injury and the relation between the porcine liver microstructure and rupture propagation in an experimental impact test. Porcine liver specimens (n = 24) were uniformly compressed using a drop tower technique and four impact heights (200, 300, 400 and 500 mm; corresponding velocities: 1.72, 2.17, 2.54 and 2.88 m s-1). The changes in intravascular pressure were measured via catheters placed in portal vein and caudate vena cava. The induced injuries were analyzed on the macroscopic level according to AAST grade and AIS severity. Rupture propagation with respect to liver microstructure was analyzed using stereological methods. MAIN RESULTS: Macroscopic ruptures affected mostly the interface between connective tissue surrounding big vessels and liver parenchyma. Histological analysis revealed that the ruptures avoided reticular fibers and interlobular septa made of connective tissue on the microscopic level. SIGNIFICANCE: The present findings can be used for evaluation of HBMs of liver behavior in impact situations.

Identification of the LLDPE Constitutive Material Model for Energy Absorption in Impact Applications.

Current industrial trends bring new challenges in energy absorbing systems. Polymer materials as the traditional packaging materials seem to be promising due to their low weight, structure, and production price. Based on the review, the linear low-density polyethylene (LLDPE) material was identified as the most promising material for absorbing impact energy. The current paper addresses the identification of the material parameters and the development of a constitutive material model to be used in future designs by virtual prototyping. The paper deals with the experimental measurement of the stress-strain relations of linear low-density polyethylene under static and dynamic loading. The quasi-static measurement was realized in two perpendicular principal directions and was supplemented by a test measurement in the 45° direction, i.e., exactly between the principal directions. The quasi-static stress-strain curves were analyzed as an initial step for dynamic strain rate-dependent material behavior. The dynamic response was tested in a drop tower using a spherical impactor hitting a flat material multi-layered specimen at two different energy levels. The strain rate-dependent material model was identified by optimizing the static material response obtained in the dynamic experiments. The material model was validated by the virtual reconstruction of the experiments and by comparing the numerical results to the experimental ones.

The histological microstructure and in vitro mechanical properties of the human female postmenopausal perineal body.

OBJECTIVE: The perineal body connects muscles from the pelvic floor and is critical for support of the lower part of the vagina and proper function of the anal canal. We determined mechanical parameters and volume fractions of main components of the human female postmenopausal perineal body. METHODS: The specimens were taken from 15 fresh female cadavers (age 74 ±â€Š10, mean ±â€Šstandard deviation). Seventy-five specimens from five regions of the perineal body were processed histologically to assess volume fractions of tissue components using stereological point testing grid. Fifteen specimens taken from the midline region were loaded uniaxially with 6 mm/min velocity until tissue rupture to determine Young's modulus of elasticity, ultimate stresses, and strains. RESULTS: The perineal body was composed of collagen (29%), adipose cells (27%), elastin (7%), smooth muscle (11%), and skeletal muscle (3%). The residual tissue (19%) constituted mostly peripheral nerves, lumina of blood vessels, fibroblasts, and fibrocytes. Young's modulus of elasticity at midline region was 18 kPa (median) at small and 232 kPa at large deformations, respectively. The ultimate stress was 172 kPa and the ultimate strain was 1.4. CONCLUSIONS: We determined the structural and mechanical parameters of the perineal body. The resultant data could be used as input for models simulating pelvic floor prolapse or dysfunction.

The histological microstructure and in vitro mechanical properties of pregnant and postmenopausal ewe perineal body.

OBJECTIVE: The mechanical properties and microstructure of the perineal body are important for the improvement of numerical models of pelvic organs. We determined the mechanical parameters and volume fractions of the ewe perineal body as an animal model. METHODS: The 39 specimens of 13 pregnant swifter ewes delivering by cesarean section (aged 2 years, weight 61.2 ±â€Š6.2 kg (mean ±â€Šstandard deviation) and 24 specimens of 8 postmenopausal swifter ewes 150 days after surgical ovariectomy (aged 7 years, 58.6 ±â€Š4.6 kg)) were loaded uniaxially to determine Young's moduli of elasticity in the small (E0) and large (E1) deformation regions, and ultimate stresses and strains. The 63 adjacent tissue samples were processed histologically to assess volume fractions of smooth and skeletal muscle, adipose cells, elastin, and type I collagen using a stereological point testing grid. We compared the structural and mechanical differences along the ewe perineal body, and between pregnant and postmenopausal groups. RESULTS: The pregnant/postmenopausal perineal body was composed of smooth muscle (12/14%; median), skeletal muscle (12/16%), collagen (10/23%), elastin (8/7%), and adipose cells (6/6%). The E0 was 37/11 kPa (median), E1 was 0.97/1.04 MPa, ultimate stress was 0.55/0.59 MPa, and ultimate strain was 0.90/0.87 for pregnant/postmenopausal perineal body. The perineal body showed a structural and mechanical stability across the sites. The pregnant ewes had a higher amount of skeletal muscle, higher E0, and a less amount of collagen when compared with postmenopausal ewes. CONCLUSIONS: The data can be used as input for models simulating vaginal delivery, pelvic floor prolapsed, or dysfunction.

Numerical and length densities of microvessels in the human brain: Correlation with preferential orientation of microvessels in the cerebral cortex, subcortical grey matter and white matter, pons and cerebellum.

To provide basic data on the local differences in density of microvessels between various parts of the human brain, including representative grey and white matter structures of the cerebral hemispheres, the brain stem and the cerebellum, we quantified the numerical density NV and the length density LV of microvessels in two human brains. We aimed to correlate the density of microvessels with previously published data on their preferential orientation (anisotropy). Microvessels were identified using immunohistochemistry for laminin in 32 samples harvested from the following brain regions of two adult individuals: the cortex of the telencephalon supplied by the anterior, middle, and posterior cerebral artery; the basal ganglia (putamen and globus pallidus); the thalamus; the subcortical white matter of the telencephalon; the internal capsule; the pons; the cerebellar cortex; and the cerebellar white matter. NV was calculated from the number of vascular branching points and their valence, which were assessed using the optical disector in 20-µm-thick sections. LV was estimated using counting frames applied to routine sections with randomized cutting planes. After correction for shrinkage, NV in the cerebral cortex was 1311±326mm-3 (mean±SD) and LV was 255±119mm-2. Similarly, in subcortical grey matter (which included the basal ganglia and thalamus), NV was 1350±445mm-3 and LV was 328±117mm-2. The vascular networks of cortical and subcortical grey matter were comparable. Their densities were greater than in the white matter, with NV=222±147mm-3 and LV=160±96mm-2. NV was moderately correlated with LV. In parts of brain with greater NV, blood vessels lacked a preferential orientation. Our data were in agreement with other studies on microvessel density focused on specific brain regions, but showed a greater variability, thus mapping the basic differences among various parts of brain. To facilitate the planning of other studies on brain vascularity and to support the development of computational models of human brain circulation based on real microvascular morphology; stereological data in form of continuous variables are made available as supplements.

Generating standardized image data for testing and calibrating quantification of volumes, surfaces, lengths, and object counts in fibrous and porous materials using X-ray microtomography.

Quantification of the structure and composition of biomaterials using micro-CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three-dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro-CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro-CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro-CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro-CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open-source software for calibrating quantitative micro-CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three-dimensional fibrillar and porous structures in micro-CT scans.

The composition and biomechanical properties of human cryopreserved aortas, pulmonary trunks, and aortic and pulmonary cusps.

Human cryopreserved allografts of pulmonary and aortic heart valves, aortas and pulmonary trunks are used for valve replacement. However, it is unknown how the composition of these allografts relate to their mechanical properties. Our aims were to correlate the histological compositions and passive mechanical properties of aortic and pulmonary valves and to observe the microcracks of aortas and pulmonary trunks. The following parameters were quantified: ultimate stress; ultimate strain; Young's modulus of elasticity; valve cusp wall thickness; pulmonary and aortic intima-media thickness; area fraction of elastin, collagen and calcification; and length density of elastic fibres. The propagation of experimentally induced microcracks avoided elastic fibres. Ultimate strain was negatively correlated with the area fraction of calcification (r=-0.4) in aortas. Ultimate stress (r=0.27) and Young's modulus in small deformation (r=0.29) and in large deformation (r=0.32) correlated with wall thickness in valve cusps. Young's modulus (r=0.34) and ultimate strain (r=0.31) correlated with intima-media thickness. Ultimate strain correlated with the area fraction of elastin (r=-0.40) and collagen in the arteries (r=0.31). As conventional histology does not fully explain the mechanical properties of cryopreserved grafts, both morphological and biomechanical tests should be used complementarily when characterizing the ageing of the grafts.

Stereological quantification of microvessels using semiautomated evaluation of X-ray microtomography of hepatic vascular corrosion casts.

PURPOSE: Quantitative description of hepatic microvascular bed could contribute to understanding perfusion CT imaging. Micro-CT is a useful method for the visualization and quantification of capillary-passable vascular corrosion casts. Our aim was to develop and validate open-source software for the statistical description of the vascular networks in micro-CT scans. METHODS: Porcine hepatic microvessels were injected with Biodur E20 resin, and the resulting corrosion casts were scanned with 1.9-4.7 [Formula: see text] resolution. The microvascular network was quantified using newly developed QuantAn software both in randomly selected volume probes (n = 10) and in arbitrarily outlined hepatic lobules (n = 4). The volumes, surfaces, lengths, and numbers of microvessel segments were estimated and validated in the same data sets with manual stereological counting. Calculations of tortuosity, radius histograms, length histograms, exports of the skeletonized vascular network into open formats, and an assessment of the degree of their anisotropy were performed. RESULTS: Within hepatic lobules, the microvessels had a volume fraction of 0.13 [Formula: see text] 0.05, surface density of 21.0 [Formula: see text] 2.0 [Formula: see text], length density of 169.0 [Formula: see text] 40.2 [Formula: see text], and numerical density of 588.5 [Formula: see text] 283.1 [Formula: see text]. Sensitivity analysis of the automatic analysis to binary opening, closing, threshold offset, and aggregation radius of branching nodes was performed. CONCLUSION: The software QuantAn and its source code are openly available to researchers working in the field of stochastic geometry of microvessels in micro-CT scans or other three-dimensional imaging methods. The implemented methods comply with reproducible stereological techniques, and they were highly consistent with manual counting. Preliminary morphometrics of the classical hepatic lobules in pig were provided.

Segmental differences in the orientation of smooth muscle cells in the tunica media of porcine aortae.

The orientation of vascular smooth muscle cells of porcine aortae was assessed to test the widely accepted assumption that these smooth muscle cells are arranged in two helices. We used tangential histological sections of 82 samples of five anatomical segments of thoracic and abdominal porcine aortae and three age groups in animals ranging in age from 5 to 210 days. The distribution of the orientation of smooth muscle cell nuclei in five proximodistal segments of the porcine aortae was determined using an algorithm that fitted a mixture of one to five von Mises probability distributions of the data retrieved from histological micrographs. Automated tracking of the nuclei was confirmed by and consistent with manual histological analysis. The orientation of the vascular smooth muscle cells was successfully fitted using two von Mises distributions in most of the samples with different ages, wall thicknesses, and anatomical positions, which corresponds to two populations of vascular smooth muscle cells. A minor fraction of samples also required a tertiary von Mises distribution to describe the orientation of the smooth muscle cell nuclei. The distribution of vascular smooth muscle cells in five aortic segments ranging from the thoracic ascending aorta to the abdominal intrarenal aorta exhibited similar main directions but different shapes. These results are consistent with the widely used model of two muscular helices intermingling in the arterial wall. Furthermore, we calculated the central angles of symmetry and the mean value of angles between the two assumed smooth muscle directions. We also successfully approximated the orientation of the smooth muscle cells using a mixture of von Mises distributions and our open-source software named dist_mixtures. This method is openly available to researchers who are interested in mathematically assessing the orientation of cell nuclei in various tissues.