Petra Kochová

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.

Elastic three-dimensional poly (ε-caprolactone) nanofibre scaffold enhances migration, proliferation and osteogenic differentiation of mesenchymal stem cells

We prepared 3D poly (ε‐caprolactone) (PCL) nanofibre scaffolds and tested their use for seeding, proliferation, differentiation and migration of mesenchymal stem cell (MSCs).

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 Youngs 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.

Optimized conditions for mesenchymal stem cells to differentiate into osteoblasts on a collagen/hydroxyapatite matrix.

Collagen/hydroxyapatite (HA) composite scaffolds are known to be suitable scaffolds for seeding with mesenchymal stem cells (MSCs) differentiated into osteoblasts and for the in vitro production of artificial bones. However, the optimal collagen/HA ratio remains unclear. Our study confirmed that a higher collagen content increased scaffold stiffness but that a greater stiffness was not sufficient for bone tissue formation, a complex process evidently also dependent on scaffold porosity. We found that the scaffold pore diameter was dependent on the concentration of collagen and HA and that it could play a key role in cell seeding. In conclusion, the optimal scaffold for new bone formation and cell proliferation was found to be a composite scaffold formed from 50 wt % HA in 0.5 wt % collagen I solution.

Collagen/hydroxyapatite scaffold enriched with polycaprolactone nanofibers, thrombocyte-rich solution and mesenchymal stem cells promotes regeneration in large bone defect in vivo

A three-dimensional scaffold of type I collagen and hydroxyapatite enriched with polycaprolactone nanofibers (Coll/HA/PCL), autologous mesenchymal stem cells (MSCs) in osteogenic media, and thrombocyte-rich solution (TRS) was an optimal implant for bone regeneration in vivo in white rabbits. Nanofibers optimized the viscoelastic properties of the Coll/HA scaffold for bone regeneration. MSCs and TRS in the composite scaffold improved bone regeneration. Three types of Coll/HA/PCL scaffold were prepared: an MSC-enriched scaffold, a TRS-enriched scaffold, and a scaffold enriched with both MSCs and TRS. These scaffolds were implanted into femoral condyle defects 6 mm in diameter and 10-mm deep. Untreated defects were used as a control. Macroscopic and histological analyses of the regenerated tissue from all groups were performed 12 weeks after implantation. The highest volume and most uniform distribution of newly formed bone occurred in defects treated with scaffolds enriched with both MSCs and TRS compared with that in defects treated with scaffolds enriched by either component alone. The modulus of elasticity in compressive testing was significantly higher in the Coll/HA/PCL scaffold than those without nanofibers. The composite Coll scaffold functionalized with PCL nanofibers and enriched with MSCs and TRS appears to be a novel treatment for bone defects.

Time-regulated drug delivery system based on coaxially incorporated platelet α-granules for biomedical use

AIM Platelet derivatives serve as an efficient source of natural growth factors. In the current study, α-granules were incorporated into coaxial nanofibers. MATERIALS & METHODS A nanofiber scaffold containing α-granules was prepared by coaxial electrospinning. The biological potential of the nanofiber scaffold was evaluated in chondrocyte and mesenchymal stem cell cultivation studies. Additionally, the concentration of TGF-β1 was determined. RESULTS Microscopy studies showed that intact α-granules were incorporated into the coaxial nanofibers. The cultivation tests showed that the novel scaffold stimulated viability and extracellular matrix production of chondrocytes and mesenchymal stem cells. In addition, the concentration of growth factors necessary for the induction of cell proliferation significantly decreased. CONCLUSION The system preserved α-granule bioactivity and stimulated cell viability and chondrogenic differentiation of mesenchymal stem cells. Core/shell nanofibers incorporating α-granules are a promising system for tissue engineering, particularly cartilage engineering.

The contribution of vascular smooth muscle, elastin and collagen on the passive mechanics of porcine carotid arteries

The main components responsible for the mechanical behavior of the arterial wall are collagen, elastin, and smooth muscle cells (SMCs) in the medial layer. We determined the structural and mechanical changes in porcine carotid arteries after administration of Triton® X-100, elastase, and collagenase using the inflation-deflation test. The arteries were intraluminarly pressurized from 0 to 200 mmHg, and the outer diameter of the artery was measured. The pressure-strain elastic modulus was determined based on the pressure/diameter ratio. The intima-media thickness, wall thickness, thickness of the tunica adventitia layer, and the area fractions of SMCs, elastin, and collagen within the arterial wall (A(A)(SMC/elastin/collagen, wall)) were measured using stereological methods. The relative changes in the relevant components of the treated samples were as follows: the decrease in A(A)(SMC, wall) after administration of Triton® X-100 was 11% ± 7%, the decrease in A(A)(elastin, wall) after administration of elastase was 40% ± 22%, and the decrease in A(A)(collagen, wall) after the application of collagenase was 51% ± 22%. The Triton® X-100 treatment led to a decrease in the SMC content that was associated with enlargement of the arterial wall (outer diameter) for pressures up to 120 mmHg, and with mechanical stiffening of the arterial wall at higher pressures. Elastase led to a decrease in the elastin content that was associated with enlargement of the arterial wall, but not with stiffening or softening. Collagenase led to a decrease in collagen content that was associated with a change in the stiffness of the arterial wall, although the exact contribution of mechanical loading and the duration of treatment (enlargement) could not be quantified.

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.

Vasa vasorum quantification in human varicose great and small saphenous veins

Recent research regarding saphenous vasa vasorum (VV) has focused on two main topics: the VV during varicogenesis in chronic venous insufficiency and the VV in saphenous grafts used in reconstructive vascular surgery. Our aim has been (i) to establish a technique for the histological quantification of the VV in human varicose great and small saphenous veins and (ii) to describe the density and distribution of the vasa vasorum within varicose veins. Great (n=11) and small (n=5) saphenous veins (length, 15-40cm) were collected from 12 patients who were undergoing venous stripping due to chronic venous insufficiency (Clinical-Etiology-Anatomy-Pathophysiology class 2-3). The veins were divided into 5-cm long segments. In total, 92 tissue blocks were collected to trace the variability of the density and distribution of the vasa vasorum in the proximo-distal direction. The endothelium was detected by immunohistochemistry using the von Willebrand factor. We quantified the number of microvessel profiles per section area and the relative distance of the microvessels from the outer border of the adventitia. The VV did not exhibit a preferential orientation in the varicose veins. VV density profiles were highest in the middle third of the venous wall and lowest in the inner third of the venous wall. Both the density and distribution of VV were uniform along the veins, and no differences were observed between the great and small saphenous veins. The VV density was statistically independent of the relative distance from the adventitia. The usability of this technique for perioperative frozen sections remains to be tested.

Quantification of compact bone microporosities in the basal and alveolar portions of the human mandible using osteocyte lacunar density and area fraction of vascular canals

Based on the orientation of the osteons, the basal portion and the alveolar portion of the body of the human mandible can be distinguished. In the compact bone, two types of microporosities can be quantified, the osteocyte lacunae and the vascular canals. Our aim was (i) to perform three-dimensional reconstruction of osteocyte lacunae to suggest an efficient means of sampling to estimate their numerical density and (ii) to compare bone microporosities in the basal and the alveolar portions of ten mandibles. Using optical disector, we estimated the density of osteocyte lacunae, and using a stereological point-counting technique, we quantified the area fraction of the vascular canals. The diameter of the lacunae was 14±3μm. While the fraction of vascular canals was comparable in both parts of the body of the mandible, the numerical density of osteocyte lacunae was higher (p=0.007) in the alveolar portion (17056±1264/mm(3)) than in the basal portion (14522±665/mm(3)). The lacunar and vascular microporosities were statistically independent of each other. As this is the first three-dimensional counting of osteocyte lacunae, we discuss the relation of this parameter to the biomechanics of the mandible, and we compare our data with previously used two-dimensional methods. We present an efficient sampling method that is useful for the histological description of bone microporosities. When taking into account the spatial characteristics of lacunae, the locally specific numerical density of lacunae can be easily assessed with the three-dimensional counting method, which is not biased by the variation in size and orientation of the lacunae.