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Development of Material Based on Hyaluronic Acid s Hydrogels for Myocardial Regeneration
Kovářová, Lenka ; Kubala,, Lukáš (referee) ; Prokš,, Vladimír (referee) ; Pekař, Miloslav (advisor)
The thesis is focused on material development based on hyaluronic acid usable in regenerative medicine, especially for heart tissue regeneration after myocardial infarction. The object of the study is the oxidized form of hyaluronic acid (HA-Ox) and hydroxyphenyl derivative of HA (HA-TA). HA-Ox can be crosslinked with a bifunctional alkoxyamine POA and HA-TA undergoes an enzymatic reaction in the presence of hydrogen peroxide catalysed by horseradish peroxidase leading to gel formation. To describe the materials, chemical and physical properties, gelation kinetics and conditions of crosslinking reactions were studied. Hydrogels were characterized by mechanical and viscoelastic properties, degradability or stability in simulated body fluids. These hydrogels serve as scaffolds for the selected cell type. To promote cell adhesion and viability, an RGD sequence has been bonded to the structure of HA-TA. This resulting material is also compatible with selected applicators. Its viscosity and extrusion force are low enough to allow application with a catheter with a very small internal diameter. The applicability of the material through the supply tube to the hydrogel reservoir of the second SPREADS device showed good homogeneity, cell distribution and viability. Finally, the material was applied in vivo using these devices during a preclinical study.
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Simulation of adhesion and transmigration imune-cells through capillary wall
Morgaenko, Katsiarina ; Čmiel, Vratislav (referee) ; Chmelíková, Larisa (advisor)
Teoretická část této práce obsahuje biofyzikální popis endoteliální vrstvy a transmigrace buněk přes tuto vrstvu. Dále popisuje charakteristiky všech důležitých komponent pro vytvoření modelu cévy in vitro (endoteliální buňky, kmenové buňky, leukocyty, Calcein AM, PKH26). Praktická část práce je věnována sestavení in-vitro modelů dvojrozměrných a trojrozměrných endoteliálních vrstev, jejich zobrazení pomoci mikroskopu a testování interakce leukocytů a kmenových buněk s těmito endoteliálními vrstvami. V závěru je navrhnut nejlepší tvar a velikost mikrofluidních systémů simulujících kapiláry.
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3D bioprinting of stem cells and analysis of microscopic images
Kandra, Mário ; Svoboda, Ondřej (referee) ; Kolářová, Jana (advisor)
In this diploma thesis we are discussing about using 3D bioprinting in tissue engineering. We are discribing using biomaterials for construction scaffolder and aplication stem cells in 3D bioprinting. Last section of theoretical part deals with very often used techniques of 3D bioprinting and we are focused on extrusion technique. In the practical part we propose a method for print vasculars structures. We realized prototype of print head, her design and 3D printing of individual parts. To mechanical part we create a control system for printing control. At the end we visualize the organization of the cells using program modules.
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In vitro models for studying Syncytin-1-induced fusion of trophoblast cells
Jech, Lukáš ; Trejbalová, Kateřina (advisor) ; Zíková, Martina (referee)
Trophoblast cell fusion is essential for human placenta development. Apart from initiating blastocyst implantation, syncytialization is critical for optimal nutrient and gas exchange between mother and fo- etus. Multicellular syncytia called syncytiotrophoblast covers the surface of the branched structure of chorionic villi, which is in direct contact with maternal blood. Impairment of the syncytialization process leads to insufficient fetal nutrition and severe pregnancy complications. Syncytia formation is induced by the interaction of the surface glycoprotein of retrovital origin, Syncytin-1, with its receptor. Despite the significance of these processes, the details of cell fusion and trophoblast differentiation remain unk- nown. Furthermore, because of its uniqueness, the human placenta cannot be covered by animal models. As a result, research into human placental development, especially Syncytin-1-induced trophoblast cell fusion, is limited to in vitro trophoblast models. These models include primary trophoblast cell cultures and trophoblast cell lines, which can be obtained by immortalizing cell cultures or extracted from trophoblast tumours. Dedifferentiated trophoblast stem cell cultures were also established. The most recent approach, however, involves the direct reprogramming of dermal...
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Potential of mesenchymal stem cells and nanoparticles in the treatment of retinal disorders
Kettner, Ondřej ; Heřmánková, Barbora (advisor) ; Filová, Eva (referee)
Retinal degenerative diseases are recognized worldwide as one of the predominant causes underlying irreversible visual impairment and even blindness. The current standard treatment has helped in both the prevention and supportive treatment of several pathologies, especially if they relate to the retina. However, many of these methods are rather invasive in terms of procedure with the subsequent possibility of infection, inflammation or even retinal detachment with the potential risk of vision loss. These restrictions represent a significant cost to patients' quality of life and also have an economic impact on the healthcare system, so new treatment options are being sought. Mesenchymal stem cells (MSCs) are a promising cell therapy tool for the treatment of many previously untreatable defects and diseases. In addition to MSCs, the use of nanoparticles and nanofibrous carriers is currently being studied in the diagnosis and therapy of retinal diseases. Combining the immunomodulatory and regenerative properties of MSCs and nanotechnologies offers a promising therapeutic strategy for a number of diseases. This thesis is therefore devoted to the current state of the use of nanomaterials, MSCs and their combination that may provide new opportunities in the treatment of retinal degenerative diseases.
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Mechanisms of Vascularization in Skin Tissue Engineering
Futóová, Terézia ; Brož, Antonín (advisor) ; Šuca, Hubert (referee)
Tissue engineering is a multidisciplinary field dealing with the fabrication of artificial tissue substitutes for regenerative medicine. Current regenerative medicine uses various types of tissue grafts, which have different advantages and disadvantages depending on their origin, such as insufficient amount of replacement tissue when using autologous grafts or immunogenicity of allogeneic or xenogeneic grafts. An alternative could be artificial tissue replacement. Artificial tissue constructs may consist of a non-living matrix and a cellular component. The cellular component may remodel the construct, form a functional part of the construct, or help integrate the construct into the host body. A significant problem in the formation of such replacements is sufficient vascularization. It is essential to keep cells in larger tissue constructs alive. Vascularization can be enhanced by the addition of vascular endothelial cells that can form capillaries independently within the construct. Vascular formation can also be aided by angiogenic growth factors by their direct application to the construct or by their formation, e.g. in stem cells cultured in the construct. Another approach is 3D bioprinting, allowing direct placement of specific cell types, growth factors or biomaterials in the construct. This...
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Differentiation of mesenchymal stem cells on fibrin assemblies supported by immobilized growth factors FGF2 and VEGF
Musílková, Jana ; Filová, Elena ; Kaplan, Ondřej ; Bačáková, Lucie
Bioartificial heart valves and vascular grafts prepared from decellularized tissues could be recellularized with bone marrow-derived mesenchymal stem cells (MSCs) that are able to differentiate into both smooth muscle cells and endothelial cells. MSCs differentiation is facilitated by sustained release of growth factors. In our study assemblies based on fibrin, fibrin with heparin, fibrin with adsorbed or covalently-immobilized vascular endothelial growth factor A165 (VEGF) or basic fibroblast growth factor (FGF-2) via binding to heparin attached to fibrin have been prepared and were evaluated for their stimulation of MSCs differentiation. We estimated the mRNA expression of endothelial marker CD31 (PECAM1), smooth muscle marker α-actin (ACTA2), osteoblast markers osteocalcin (BGLAP) and alkaline phosphatase (ALP). The gene expression was estimated using RT-PCR on days 1, 7 and 21 after seeding. The cell morphology and viability was evaluated by LIVE/DEAD staining. VEGF, both adsorbed and covalently bound, increased significantly the expression of smooth muscle marker α-actin. The mRNA expression of ACTA2 on day 7 and 21 raised more than 200 times in comparison to control samples (undifferentiated cells before seeding). The ACTA2 gene expression significantly exceeded the expression of all other evaluated genes at all time intervals. Moreover, on day 21, the late smooth muscle marker desmin (DES) was steeply rising in cells cultivated on assemblies containing heparin and covalently bound VEGF. The expression of osteocalcin was minimal. We conclude that fibrin assembly containing covalently bound VEGF is the most convenient for MSCs differentiation towards smooth muscle cells.
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Awareness of pregnant women about cord blood donation
KOLMANOVÁ, Michaela
This bachelor's thesis is dedicated to donating umbilical cord blood. The thesis consists of a theoretical and practical part. The theoretical part contains a description of the placenta and its functions, umbilical cord blood, stem cells. Furthermore, the theoretical part contains a description of the technique of cord blood collection, contraindications of the collection and processing of umbilical cord blood. It also describes the education of midwives on this issue. Umbilical cord blood banks and legislative provisions are listed here. The aim of the bachelor's thesis was to find out how knowledgeable pregnant women are about this issue. A quantitative research survey was used in the practical part of this work. The questionnaire created contained 18 closed questions. The research team was pregnant women. This set consisted of 244 respondents. Of these, 155 (64%) first-time parents 89 (36%) multi-parents. Of these, 41.39% of respondents know the use of umbilical cord blood. Only 18.03% of respondents know what they need to do to be able to take umbilical cord blood. 19.67% of respondents reported the correct time of collection of umbilical cord blood. 13.52% of respondents reported that the maximum storage period of donated umbilical cord blood is 20 years. Two hypotheses were established in this work. The first hypothesis focused on whether pregnant women are given more information about this issue by a midwife or gynecologist. 5.70% of respondents received information from a midwife. 3.70% of respondents received information from a gynecologist. Statistical processing of the hypothesis has shown us that there is no difference in who gives information. Women are informed from the midwife as well as from the gynecologist. The second hypothesis was whether multi-parents are more informed about umbilical cord blood donation than first-time parents. Multi-parents accounted for 36%, and first-time parents 64%. 46.06% of multi-parents and 38.71% of first-time parents said they knew the use of umbilical cord blood. 20.22% of multi-parents and 19.35% of first-time parents know the time of collection of umbilical cord blood. 50.56% of multi-parents and 39.35% of first-time parents reported that the donation was not risky for newborns. We confirmed the second hypothesis. The results of this work show that women have very little information on the issue of umbilical cord blood donation. The midwife is a competent person, so women could be more education about this issue. This work can be used in seminars for midwives.
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Research of epigenetic aspects of hematopoietic and spermatogenesis stem cells.
Hybešová, Michaela ; Pimková, Kristýna (advisor) ; Děd, Lukáš (referee)
Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...
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Stimulation of mesenchymal stem cells osteogenic differentiation using perfusion bioreactor
Šljivnjak, Erna ; Rampichová, Michala (advisor) ; Rösel, Daniel (referee)
Bone cells in vivo reside in a dynamic environment exposed to constant chemical and mechanical stimuli caused by the interstitial fluid flow. It is hypothesized that perfusion of the medium through the scaffold increases the mass transport and creates at the same time shear stress, thereby in vitro simulating the interstitial fluid effects and bone tissue formation conditions. This work examined the effects of perfusion flow rates on cell viability, proliferation, migration and osteogenic differentiation of human mesenchymal stem cells within cell-seeded 3D poly-ε-caprolactone scaffolds. Scaffolds were perfused for 21 days at flow rates 1, 3 and 5 mL/min and were compared to the scaffolds from static culture. Cells were most viable, had upregulated expression of osteogenic markers collagen type I and highest alkaline phosphatase activity under flow rate 1 mL/min when compared to their static counterparts. Cells proliferated the most under flow rate 3 mL/min when compared to their static counterparts. Flow rate 5 mL/min did not significantly differ from the static culture in any of the examined parameters. Cell migration into the scaffold was not improved upon exposure to perfusion. This data confirms that medium perfusion may benefit cell proliferation and osteogenic differentiation by enhancing...
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