Tereza Kubíková

Segmental and age differences in the elastin network, collagen, and smooth muscle phenotype in the tunica media of the porcine aorta

The porcine aorta is often used in studies on morphology, pathology, transplantation surgery, vascular and endovascular surgery, and biomechanics of the large arteries. Using quantitative histology and stereology, we estimated the area fraction of elastin, collagen, alpha-smooth muscle actin, vimentin, and desmin within the tunica media in 123 tissue samples collected from five segments (thoracic ascending aorta; aortic arch; thoracic descending aorta; suprarenal abdominal aorta; and infrarenal abdominal aorta) of porcine aortae from growing domestic pigs (n=25), ranging in age from 0 to 230 days. The descending thoracic aorta had the greatest elastin fraction, which decreased proximally toward the aortic arch as well as distally toward the abdominal aorta. Abdominal aortic segments had the highest fraction of actin, desmin, and vimentin positivity and all of these vascular smooth muscle markers were lower in the thoracic aortic segments. No quantitative differences were found when comparing the suprarenal abdominal segments with the infrarenal abdominal segments. The area fraction of actin within the media was comparable in all age groups and it was proportional to the postnatal growth. Thicker aortic segments had more elastin and collagen with fewer contractile cells. The collagen fraction decreased from ascending aorta and aortic arch toward the descending aorta. By revealing the variability of the quantitative composition of the porcine aorta, the results are suitable for planning experiments with the porcine aorta as a model, i.e. power test analyses and estimating the number of samples necessary to achieving a desirable level of precision. The complete primary morphometric data, in the form of continuous variables, are made publicly available for biomechanical modeling of site-dependent distensibility and compliance of the porcine aorta.

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.

The combination of nanofibrous and microfibrous materials for enhancement of cell infiltration and in vivo bone tissue formation

Fibrous scaffolds are desired in tissue engineering applications for their ability to mimic extracellular matrix. In this study we compared fibrous scaffolds prepared from polycaprolactone using three different fabrication methods, electrospinning (ES), electro-blowing and melt-blown combined with ES. Scaffolds differed in morphology, fiber diameters and pore sizes. Mesenchymal stem cell adhesion, proliferation and osteogenic differentiation on scaffolds was evaluated. The most promising scaffold was shown to be melt-blown in combination with ES which combined properties of both technologies. Microfibers enabled good cell infiltration and nanofibers enhanced cell adhesion. This scaffold was used for further testing in critical sized defects in rabbits. New bone tissue formation occurred from the side of the treated defects, compared to a control group where only fat tissue was present. Polycaprolactone fibrous scaffold prepared using a combination of melt-blown and ES technology seems to be promising for bone regeneration. The practical application of results is connected with enormous production capacity and low cost of materials produced by melt-blown technology, compared to other bone scaffold fabrication methods.

Stereological analysis of size and density of hepatocytes in the porcine liver

The porcine liver is frequently used as a large animal model for verification of surgical techniques, as well as experimental therapies. Often, a histological evaluation is required that include measurements of the size, nuclearity or density of hepatocytes. Our aims were to assess the mean number‐weighted volume of hepatocytes, the numerical density of hepatocytes, and the fraction of binuclear hepatocytes (BnHEP) in the porcine liver, and compare the distribution of these parameters among hepatic lobes and macroscopic regions of interest (ROIs) with different positions related to the liver vasculature. Using disector and nucleator as design‐based stereological methods, the morphometry of hepatocytes was quantified in seven healthy piglets. The samples were obtained from all six hepatic lobes and three ROIs (peripheral, paracaval and paraportal) within each lobe. Histological sections (thickness 16 μm) of formalin‐fixed paraffin‐embedded material were stained with the periodic acid‐Schiff reaction to indicate the cell outlines and were assessed in a series of 3‐μm‐thick optical sections. The mean number‐weighted volume of mononuclear hepatocytes (MnHEP) in all samples was 3670 ± 805 μm3 (mean ± SD). The mean number‐weighted volume of BnHEP was 7050 ± 2550 μm3. The fraction of BnHEP was 4 ± 2%. The numerical density of all hepatocytes was 146 997 ± 15 738 cells mm−3 of liver parenchyma. The porcine hepatic lobes contained hepatocytes of a comparable size, nuclearity and density. No significant differences were identified between the lobes. The peripheral ROIs of the hepatic lobes contained the largest MnHEP with the smallest numerical density. The distribution of a larger MnHEP was correlated with a larger volume of BnHEP and a smaller numerical density of all hepatocytes. Practical recommendations for designing studies that involve stereological evaluations of the size, nuclearity and density of hepatocytes in porcine liver are provided.

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; Youngs 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 Youngs modulus in small deformation (r=0.29) and in large deformation (r=0.32) correlated with wall thickness in valve cusps. Youngs 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.

Histological Composition and Mechanical Properties of Cryopreserved Samples of Aortic and Pulmonary Valves

Human cryopreserved allografts of pulmonary and aortic valves are routinely used as total valve replacement. For successful surgery it is needed to sufficiently preserve biomechanical properties and histological structures of allografts. However, it is not known how the mechanical properties of these allografts relate to their histological composition. The aim of our study was to compare the histological composition and mechanical properties of the valves. From allografts we prepared 2 valve cusps and samples of aorta or pulmonary trunk. In a previous study we had measured following parameters: ultimate stress, ultimate strain, Young’s moduli of elasticity, intima-media thickness, wall thickness, area fraction of elastin and area fraction of collagen in the whole wall. We found weak positive correlation between ultimate stress and Young’s modulus in small and large deformation with wall thickness in the valve cusps. In the arteries we found positive correlation between Young’s modulus in large deformation with intima-media thickness and ultimate strain with intima-media thickness and area fraction of collagen, and negative correlation between ultimate strain with area fraction of elastin. In our study we quantified also the other components of wall with mechanical significance, such as the fraction of smooth muscle cells and chondroitin sulfate, which belong to glycosaminoglycans. We did not find correlation between these components and mechanical properties of these valves. Therefore, it is recommended to perform both mechanical and histological analysis to further characterize cryopreserved allografts.

IDH1 mutation is associated with lower expression of VEGF but not microvessel formation in glioblastoma multiforme

Introduction Glioblastoma multiforme (GBM) represents the most malignant primary brain tumor characterized by pathological vascularization. Mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) were observed in GBM. We aimed to assess the intra-tumor hypoxia, angiogenesis and microvessel formation in GBM and to find their associations with IDH1 mutation status and patients prognosis. Methods 52 patients with a diagnosis of GBM were included into the study. IDH1 R132H mutation was assessed by RT-PCR from FFPE tumor samples obtained during surgery. The expression of markers of hypoxia (HIF2α), angiogenesis (VEGF), tumor microvascularity (CD31, CD34, vWF, CD105), and proliferation (Ki-67) were assessed immunohistochemically (IHC). IDH1 mutation and IHC markers were correlated with the patient survival. Results 20 from 52 GBM tumor samples comprised IDH1 R132H mutation (38.5%). The majority of mutated tumors were classified as secondary glioblastomas (89.9%). Patients with IDH1 mutated tumors experienced better progression-free survival (P = 0.037) as well as overall survival (P = 0.035) compared with wild type tumors. The significantly lower expression of VEGF was observed in GBM with IDH1 mutation than in wild type tumors (P = 0.01). No such association was found for microvascular markers. The increased expression of newly-formed microvessels (ratio CD105/CD31) in tumor samples was associated with worse patient’s progression-free survival (P = 0.026). Summary No increase in HIF/VEGF-mediated angiogenesis was observed in IDH1-mutated GBM compared with IDH1 wild type tumors. The histological assessment of the portion of newly-formed microvessels in tumor tissue can be used for the prediction of GBM patient’s prognosis.

Comparison of ground sections, paraffin sections and micro-CT imaging of bone from the epiphysis of the porcine femur for morphometric evaluation

The aim of this study was to compare data on the volume fraction of bone and the thickness of the cortical compact bone acquired during microcomputed tomography (micro-CT) analysis with data acquired from identical samples using stereological analysis of either decalcified paraffin sections or ground sections. Additionally, we aimed to compare adjacent tissue samples taken from the major trochanter of the porcine femur to map the basic biological variability of trabecular bone. Fifteen pairs of adjacent tissue blocks were removed from the major trochanter of the proximal epiphyses of porcine femurs (female pigs aged 24-39 months, weight=59.16±8.15kg). In each sample, the volume of the cortical compact bone, the volume of the trabecular bone, and the thickness of the cortical compact bone was assessed using micro-CT. Afterwards, half of the samples were decalcified and processed using paraffin histological sections. Another half was processed into ground sections. The volume and thickness of bone was assessed in histological sections using stereological techniques. There were no significant differences in the bone volumes and thicknesses measured by micro-CT and the corresponding values quantified in decalcified sections. Similarly, there were no differences between the results from micro-CT and the analysis of the corresponding ground sections. Histomorphometric studies based on relatively low numbers of undecalcified ground sections or demineralized paraffin sections of bone yield data on bone volume and the thickness of cortical compact bone that is comparable with three-dimensional micro-CT examination. The pilot data on the variability of cortical compact bone and trabecular bone volumes in the porcine major trochanter provided in this study aim for planning experiments in the field of bone healing and implantology.

Links between the Orientation of Vascular Smooth Muscle and Microscopical Composition of Aortic Segments

We analyzed histological data statistically describing the distribution of orientations of vascular smooth muscle cells (VSMC) within porcine aorta. The data were correlated with the fractions of actin, desmin, vimentin, elastin and collagen within the same samples. In samples with more contractile VSMC and less elastin, the symmetrical helices of VSMC were arranged closely to each other and they were more concentrated than in samples with fewer actin-and desmin-positive VSMC and more elastin. The findings are suitable for microstructurally-motivated biomechanical modeling of porcine aorta under normal conditions.