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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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Analysis of adherent cells confluency in 2D culture
Bracková, Michaela ; Čmiel, Vratislav (referee) ; Chmelíková, Larisa (advisor)
First part of this semester work dealing with the theoretical description of adherens cells, particularly structure and functions of this cells. Subsequently work is devoted to description of cell culturing, with mention of conditions which are necessary for cell culturing, following by substances which promote cell growth. Last part of theoretical research is concern with microscopy technique that is suitable for studying of adherent cells. Subsequently it is about fluorescence and confocal microscopes. Practical part dealing with cell culturing of adherent cells and the evaluation of realized experiments.
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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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Interactions of skin and stem cells with polymer nanofibres for construction of skin substitutes
Tomšů, Júlia ; Bačáková, Lucie (advisor) ; Sedmera, David (referee) ; Jendelová, Pavla (referee)
The skin is the largest organ of a human body with a crucial role in the maintenance of homeostasis; therefore any extensive skin injury leads to severe complications. Since the application of auto-, allo- and xeno-grafts is accompanied by severe problems like the source limitation and the graft rejection, a bioengineered skin substitute seems to be one of the promising healing approach. This work is focused mainly on the construction of a pre- vascularized skin substitute consisting of a collagen hydrogel reinforced by a biodegradable nanofibrous membrane. Another strategy described in this work is the development of temporary cellulose-based wound dressings. For both research strategies, various cell types were utilized, i.e. normal human dermal fibroblasts (NHDFs), human keratinocytes (hKs), adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). In order to enhance the cell adhesion and growth, the synthetic nanofibrous membranes were improved by protein nanocoatings. It was found out that NHDFs and ADSCs preferred fibrin nanocoatings, mainly thin fibrin homogeneous mesh on the surface of the membrane. Keratinocytes rather adhered and stratified on collagen substrates. These observations further motivated the construction of the bi-layered construct, where...
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Computational Simulation of Mechanical Behaviour of Endothelial Cells
Jakka, Veera Venkata Satya ; Gumulec, Jaromír (referee) ; Majer, Zdeněk (referee) ; Matsumoto,, Takeo (referee) ; Burša, Jiří (advisor)
Ateroskleróza je v rozvinutém světě hlavní příčinou úmrtí a finančně zatěžuje zdravotnické systémy po celém světě. Převládající hemodynamické působení spolu s lokální koncentrací mechanického napětí hrají důležitou roli v lokální povaze aterosklerózy a jejím rozvoji ve specifických oblastech lidských cév. Endotel v krevních cévách je tvořen tenkou vrstvou buněk, ležící na rozhraní mezi krevním řečištěm a cévní stěnou. Dysfunkce endoteliálních buněk se podílí na hlavních patologiích. Například ateroskleróza se rozvíjí, když jsou narušeny bariérové a protizánětlivé funkce endotelu, což umožňuje akumulaci cholesterolu a dalších materiálů v arteriální stěně. U rakoviny je klíčovým krokem v růstu nádoru jeho vaskularizace a proces migrace endoteliálních buněk. Mechanické zatížení endoteliálních buněk hraje klíčovou roli v jejich funkci a dysfunkci. Počítačové modelování může zlepšit porozumění buněčné mechanice a tím přispět k poznání vztahů mezi strukturou a funkcí různých typů buněk v různých stavech. K dosažení tohoto cíle jsou v této práci navrženy konečnoprvkové modely endoteliálních buněk, tj. model buněk plovoucích v roztoku a model buněk přilnutých k podložce, které objasňují reakci buňky na globální mechanické zatížení, jako je tah a tlak, jakož i model buňky s jeho přirozeným tvarem uvnitř endoteliální vrstvy. Zachovávají hlavní principy tensegritních struktur, jako je předpětí a spolupůsobení jednotlivých součástí, ale prvky se mohou organizovat vzájemně nezávisle. Při implementaci nedávno navržené bendo-tensegritní koncepce uvažují tyto modely namáhání mikrotubulů nejen v tahu/tlaku, ale i ohybu a také zohledňují vlnitost intermediálních filament. Modely umožňují, že jednotlivé komponenty cytoskeletu mohou změnit svůj tvar a uspořádání bez zhroucení celé buněčné struktury, dokonce i když jsou odstraněny, a umožňují nám tak vyhodnotit mechanický přínos jednotlivých cytoskeletálních složek k buněčné mechanice. Navržené modely jsou validovány porovnáním jejich křivek síla-posunutí s experimentálními výsledky. Model plovoucí buňky realisticky popisuje silově-deformační odezvu buňky při tahu a tlaku a obě reakce ilustrují nelineární zvýšení tuhosti s mechanickým zatížením. Je simulována také tlaková zkouška ploché endoteliální buňky a porovnána s testem přilnuté buňky a jeho simulací. Poté se simuluje smykový test ploché buňky, aby se vyhodnotilo její chování při smykovém zatížení vyskytujícím se v cévní stěně v důsledku proudění krve. Poté byla zkoumána mechanická odezva ploché buňky ve vrstvě endotelu za fyziologických podmínek v arteriální stěně. Později byla zkoumána buněčná odezva při odtrhování od položky během cyklických úseků pomocí 3D simulací metodou konečných prvků. Navrhované modely poskytují cenné poznatky o vzájemných souvislostech mechanických vlastností buněk, o mechanické roli jednotlivých cytoskeletálních složek i jejich synergii a o deformaci jádra za různých podmínek mechanického zatížení. Proto by práce měla přispět k lepšímu pochopení cytoskeletální mechaniky, zodpovědné za chování buněk, což může zase pomoci při zkoumání různých patologických stavů souvisejících s buněčnou mechanikou, jako je rakovina a vaskulární onemocnění.
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Vascular Endothelial Growth Factor Expression and its Application in Vascular Tissue Engineering
Mikulová, Barbora ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
This paper deals with the expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and its use in tissue engineering of vascular wall. During the work interaction of endothelial cells with the modified fibrin-based biomaterial into which vascular endothelial growth factor (VEGF-A121) has been incorporated was monitored. This modification supported the adhesion and growth of endothelial cells. Vascular endothelial growth factor VEGF-A121 is signal glycoprotein that activates transmembrane receptors on endothelial cells. VEGF-A121 is a key regulator in vasculogenesis, angiogenesis, proliferation, migration and survival of endothelial cells. In this work, this protein was heterologously expressed at a thioredoxin fusion partner in an expression system of E. coli Origami B (DE3). Recombinant VEGF-A121 was additionally coexpressed with bacterial chaperones GroEL/GroES for potential increase of its solubility and biological activity. In the next part of this work thin fibrin network was prepared by catalytic action of thrombin on the polystyrene-bound monolayer of fibrinogen. This network has been further enriched by vascular endothelial growth factor (VEGF-A121), which was covalently incorporated in it by enzyme activity of transglutaminase (factor XIIIa). The last...
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Application of nanofiber scaffolds for vesel regeneration
Bezděková, Dagmar ; Amler, Evžen (advisor) ; Holzerová, Kristýna (referee)
Although plenty of systems for vessel regeneration have been developed, no system is successful in small diameter (under 6 mm) vessel replacement yet. Synthetic materials, such as Dacron and ePTFE, have good results in large vessels replacement, but they cause thrombosis in small vessels. In addition, they are not degradable and do not allow a natural remodeling of the vessel system. Furthemore, endothelial cells, which are essential for creating natural antithrombogenic endothelium, do not adhere on these materials, as well as smooth muscle cells. Decellularized xenogenic material is the non-synthetic alternative for vessel regeneration. Appropriate detergent removes donor's cells and only extracellular matrix remains, which is able to host acceptor's cells. The main disadvantages of this system are difficulties with animal's nurture and structure violations after detergent is used. It appears that electrospun materials are the best alternative. The relatively simple process can be modified in many ways and provides then a scaffold, which mimics extracellular matrix. A big advantage of this process is the possibility to incorporate bioactive substances into a fiber. The substances serve there as an attractant for blood cells or as an anticoagulation factor. In combination with the progenitor cells seems...
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Impact of mesenchymal stem cells on islets revascularization after transplantation into the extracellular matrix
Hudzieczková, Aneta ; Girman, Peter (advisor) ; Černý, Jan (referee)
Pancreatic transplantation is the only possible treatment to induce independence from exogenous insulin administration in type 1 diabetes mellitus. However, the shortage of donor organs remains the main limitation of pancreas transplantations. The goal of the research is the preparation of a bioartificial organ based on cell therapy. Parts of the extracellular matrix obtained by decellularization of the pancreas are used for its preparation. The protein scaffolds prepared in this way are then repopulated by different cell types again. The extracellular matrix provides structural support to cells, mediates signaling for differentiation, proliferation or migration. Mesenchymal stromal cells are used in clinical therapy, have a positive effect on tissue regeneration processes, modulating the function of the extracellular matrix, suppress inflammation and promote angiogenesis. After pancreas decellularization, we repopulated the extracellular matrix with islets, mesenchymal cells and endothelial cells. Then, the pancreas was transplanted subcutaneously into syngeneic diabetic rats to observe islet revascularization. Based on sections of explanted scaffolds, we found out that revascularization of the islets was higher without the endothelial cells in the transplanted extracellular matrix. Key words:...
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Vascular Endothelial Growth Factor Expression and its Application in Vascular Tissue Engineering
Mikulová, Barbora
This paper deals with the expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and its use in tissue engineering of vascular wall. During the work interaction of endothelial cells with the modified fibrin-based biomaterial into which vascular endothelial growth factor (VEGF-A121) has been incorporated was monitored. This modification supported the adhesion and growth of endothelial cells. Vascular endothelial growth factor VEGF-A121 is signal glycoprotein that activates transmembrane receptors on endothelial cells. VEGF-A121 is a key regulator in vasculogenesis, angiogenesis, proliferation, migration and survival of endothelial cells. In this work, this protein was heterologously expressed at a thioredoxin fusion partner in an expression system of E. coli Origami B (DE3). Recombinant VEGF-A121 was additionally coexpressed with bacterial chaperones GroEL/GroES for potential increase of its solubility and biological activity. In the next part of this work thin fibrin network was prepared by catalytic action of thrombin on the polystyrene-bound monolayer of fibrinogen. This network has been further enriched by vascular endothelial growth factor (VEGF-A121), which was covalently incorporated in it by enzyme activity of transglutaminase (factor XIIIa). The last...
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Effect of soluble endoglin to endothelial cells in vitro
Klingová, Rebeka ; Fikrová, Petra (advisor) ; Doleželová, Eva (referee)
Charles Univerzity in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Rebeka Klingová Supervisor: PharmDr. Petra Fikrová, Ph.D. Title of diploma thesis: Effect of soluble endoglin to endothelial cells in vitro Aim: We determined the effects of soluble endoglin on endothelial cells, by the means of inflammatory markers. The aim of the study was to point out the possible association of the soluble endoglin with endothelial dysfunction. Methods: For our study we have selected the human endothelial cells from umbilical vein - HUVEC. We have influenced the cells with soluble endoglin in two concentrations and at two different time intervals. The results were evaluated in the statistical program, in which we have compared the control group with influenced cells and two concentrations between each other. The expressions of inflammatory markers were analyzed on the level of mRNA, using the real-time PCR method. Results: Significant changes in the expression of markers were observed on the vascular and intracellular adhesion molecule at both concentrations compared to the control group. Increased values of transcription were available also for cyclooxygenase 2 and decreased values for cadherin 5 compared to the control group. Conclusion: Changes in...
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