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Smart biomaterials for connective tissues regeneration
Hefka Blahnová, Veronika ; Filová, Eva (advisor) ; Maxová, Hana (referee) ; Motlík, Jan (referee)
Connective tissues are characterized by significant volume of extracellular matrix. Their main role is to provide a mechanical support and protection to other body organs. This thesis is focused on regeneration of bone, cartilage and osteochondral defect. In the experimental part we observed viability and differentiation of human mesenchymal stem cells. In vitro we evaluated the potential of PCL scaffold with addition of growth factors, bone xenograft with biomimetic peptides, collagen I composite with bioceramics and a titanium alloy with nanostructured surface. During following in vivo study, we implanted a cell-free scaffold made of PCL, calcium phosphate and IGF-1, bFGF, TGFβ1 and BMP-2 to osteochondral defect. Unfortunately, addition of growth factors resulted in pathological inflammatory process despite clear beneficial effect in vitro. Likewise, the biomimetic peptide sequences promoted osteogenic differentiation of human mesenchymal stem cells. Addition of certain bioceramics influenced the scaffold morphology in the manner of pore size. However, we did not observe any effect of the surface characteristics on cell behavior. The cells were influenced rather by certain material. On the other hand, surface modification of titanium scaffold by anodic oxidation revealed that the most suitable...
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The effect of nanoparticles on functional and regenerative properties of mesenchymal stem cells
Dostálová, Dominika ; Rössner, Pavel (advisor) ; Grobárová, Valéria (referee)
Mesenchymal stem cells (MSCs) represent a type of stem cells, localized in various tissues, where they are involved in the regeneration process. Because of their properties, MSC are tested for cell-based therapy. However, the treatment of injuries is often associated with bacterial infections, which complicate the process of healing. To establish a sterile environment, metallic nanoparticles (NPs) with proven antibacterial properties can be applied, for improving the healing process. Mechanisms of the antibacterial effect of NPs are non-specific and may cause the damage of eukaryotic cells. The aim of this thesis was to evaluate the effect of three antibacterial metallic NPs (silver, copper oxide and titanium dioxide) on the functional and regenerative properties of MSCs. The effect of NPs on the metabolic activity, differentiational potential, expression of genes for immunoregulatory molecules and production of cytokines by MSCs was determined. Additionally, the immunoregulatory properties of MSCs after cultivation with NPs were tested. The results showed that the metabolic activity was reduced in the presence of silver and copper oxide NPs, with the decrease dependent on the type of the tested NPs. The expression of selected immunoregulatory molecules was inhibited and the immunoregulatory...
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Mitochondrial transfer-mediated modulatory action of stem cells on immune cells
Somova, Veronika ; Krulová, Magdaléna (advisor) ; Balounová, Jana (referee)
Stem cells use different mechanisms of intercellular communication to modulate an immune response. Mitochondrial transfer is one of the mechanisms which induce metabolic changes, support cell survival, and change the phenotype of immune cells. Nevertheless, little is known about the mechanism used for transfer of mitochondria between different cell populations and the faith of mitochondria inside the acceptor cell. This thesis aims to describe the mechanism of transfer and the provided modulation. Factors that could affect mitochondrial transfer including reactive oxygen species production, apoptosis and mitochondria function were analyzed. And the impact of mitochondrial transfer on cell survival and mitophagy was described. The next aim was to compare the ability of mesenchymal stem cells (MSC) and Sertoli cells (SC) to transfer mitochondria, with MSC being more productive in the transfer of mitochondria than SC. Significant differences in the presence of mitochondria from donor MSC or SC in individual populations of immune cells were also detected. To explain these findings, the impact of reactive oxygen species on the transfer of mitochondria was analyzed in detail, although it wasn't confirmed. However, it needs to be highlighted that mitophagy plays an important role before and after...
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Importance of glycolysis and oxidative phosphorylation in the metabolism of mesenchymal stem cells
Fráňová, Markéta ; Krulová, Magdaléna (advisor) ; Rohlenová, Kateřina (referee)
Mesenchymal stem cells (MSCs) are classified as multipotent stem cells. They possess the ability to differentiate into many cell types, promote angiogenesis, increase cell survival in damaged tissue and modulate the immune response. These functions of MSCs are used in the treatment of various injuries and some diseases. This work characterizes MSCs, with a focus on their energy metabolism, specifically on the switch in their metabolic phenotype between glycolysis and oxidative phosphorylation in different states of MSCs, during cell culture and after transplantation. Finally, two modulations of MSC metabolism are presented, including cultivation in a hypoxic environment and quiescence induced by serum deprivation, which increase cell survival under the ischemic conditions that MSCs enter after transplantation. Key words: mesenchymal stem cells, metabolism, glycolysis, oxidative phosphorylation
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Fluorescent Methods in Research of Eukaryotic Cells
Chmelíková, Larisa ; Babula, Petr (referee) ; Pešl,, Martin (referee) ; Provazník, Ivo (advisor)
Tato práce zkoumá aplikaci fluorescenčních metod používaných v in vitro studiích v oblasti regenerace srdeční tkáně. Konfokální fluorescenční mikroskopie je vhodnou mikroskopickou technikou pro výzkum v této oblasti, protože umožňuje vizualizaci 3D struktur a distribuce buněk ve 3D modelech. Používané fluorescenční markery by měly být dlouhodobě stabilní, biokompatibilní a netoxické pro živé buňky. V současné době je použití nanočástic jako superparamagnetické nanočástice oxidu železa (SPION) velmi populární; velké množství studií ukazuje, že jsou vhodné pro dlouhodobé experimenty. Tento výzkum využívá superparamagnetické maghemitové nanočástice svázaným rhodaminem na jejich povrchu (SAMN-R) a popisuje jejích excitační a emisní spektrum, velikost a lokalizaci vbuňkách. Stanovení toxicity bylo provedeno měřením reaktivních forem kyslíku (ROS) a nekvantitativním měřením pomocí fluorescenční mikroskopie bylo zjištěno, že hodnota dávky 20 µg·cm-2 je optimální pro aplikaci na živé buňky. Dále byl zkoumán vliv aplikace SAMN-R na buněčnou proliferaci a motilitu, kdy ve studii buněčné proliferace a scratch assay byla použita buněčná linie fibroblastů 3T3. Poté byla studována migrace jednotlivých buněk s použitím mezenchymálních kmenových buněk (MSCs), izolovaných zlidské tukové tkáně. Následná statistická analýza nepotvrdila, že by aplikace SAMN-R měla významný vliv na buněčnou proliferaci, kolektivní migraci nebo na migraci jednotlivých buněk. Lze tedy předpokládat, že SAMN-R jsou vhodným fluorescenčním markerem pro výzkum živých buněk, včetně experimentů voblasti regenerace tkáně. MSC buňky izolované z tukové tkáně mají velký potenciál v regeneraci srdeční tkáně. Jejich interakce s buněčnou linií srdečních svalových buněk HL-1 byly studovány pomocí scratch assay, kdy se tento model jeví jako nadějný a vhodný pro studium buněčných kontaktů a jejich roli přiregeneraci buněk.
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Immunomodulatory properties of mesenchymal stem cells - use in therapy
Pavlíková, Michaela ; Krulová, Magdaléna (advisor) ; Stříž, Ilja (referee)
Mesenchymal stem cells (MSC) are extensively studied mainly due to their feasible clinical application. Therapeutic potential of MSC consists not only of the ability to differentiate into mesenchymal cells, ectodermal and endodermal cell lines, but primarily in their immunomodulatory functions. Due to their effect on immune cells, MSC promote the shift of the inflammatory immune response to antiinflammatory. The ability to suppress inflammation, together with their differentiation potential and antiapoptotic potential on the surrounding cells makes MSC a promising tool for treating serious diseases. This work discusses the effect of MSC on the individual cells of the immune system. It focuses on the description of the effect of MSC in four model cases. These are an experimental autoimmune encephalomyelitis, myocardial infarction, diabetes mellitus and skin graft transplantation. The knowledge of the mechanisms of the interactions between MSC and the cells of the immune system, together with the understanding the effect of specific conditions on MSC is essential for their use in clinical therapy. Keywords: mesenchymal stem cells, immunomodulation, autoimmune diseases, transplantation
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Immunomodulatory and regenerative potential of mesenchymal stem cells in the treatment of degenerative retinal disorders in mice
Palacká, Kateřina ; Heřmánková, Barbora (advisor) ; Grobárová, Valéria (referee)
Retinal degenerative disease are the leading cause of vision loss in adult patients. Important role in the development of these types of disease play gradual death of retinal cells and an inflammatory reaction that leads to the production of cytokines, formation of inflamasome, increased angiogenesis and scar formation. These pathologies result in irreversible degeneration of retinal cells. Retinal pigmenetd epithelium cells, photoreceptors and ganglion cells are the most frequently damaged cell types in this conditions. The available treatments are currently very limited and effective only at the early stages of the disease. Therefore, the use of stem cell- based therapy could be a promising option. For therapeutic purposes it would be possible to use mesenchymal stem cells (MSCs) which may be isolated for example from bone marrow or adipose tissue. MSCs are capable of production of neuroprotective factors, differentiation into the variety of cells types and regulation of immune response. In this study we tested the therapeutic potential of MSC administered locally to the damaged retina in an experimental model of retinal degeneration. We focused on the protective effect of MSCs on photoreceptor cells, regulation of the local immune response and expression of genes for cytokines involved in...
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Study of the effect of mesenchymal stem cells in combination with immunosuppressive therapy on inflammatory response in in vivo model
Jabůrek, Filip ; Krulová, Magdaléna (advisor) ; Brdička, Tomáš (referee)
Immunosuppressive drugs have been used for many years for the treatment of autoimmune diseases and post-transplantation treatment. While these drugs have a lot of advantages, they also show several undesirable side effects. The most common side effects are higher blood pressure, lowered renal function and susceptibility to infections. Therefore, in recent years there has been a demand for other medical approaches that do not exhibit the above-mentioned adverse effects. Among one of the newly tested approaches is the application of mesenchymal stem cells (MSCs), which possess several advantages such as immunomodulatory abilities, safety and relatively easy isolation, however, stem cell use alone has not yet provided sufficiently strong immunomodulation. Only a small part of research of MSCs is focused on their use in the combination with immunosuppressive therapy. Therefore, in my thesis I focused on the model which allows to reduce the dose of immunosuppressive drugs in the combination with MSCs. Combined therapy is more advantageous than both monotherapies thanks to lower dosages of these drugs used. It enables to decrease negative side effects of immunosuppressive drugs, when combined with MSCs to provide sufficient immunomodulation in comparison to classical therapy. The aim of my work was to...
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Mesenchymal stem cells for treatment of spinal cord injury
Dubišová, Jana ; Kubinová, Šárka (advisor) ; Hock, Miroslav (referee)
Poranenie miechy a jeho terapia pomocou mezenchymálnych kmeňových buniek Abstract Patients with serious spinal cord injury (SCI) have a little chance of a full return to their original life. This kind of injuries leaves lifelong consequences as on the patients themselves, as well as their surroundings and family. Considerable financial resources are, worldwide, granted for reintegratation of these patients into society. This work focuses on the description of spinal cord injury, its complexity and the possibility of treatment by new therapeutic methods of mesenchymal cells. The first section discusses the severity of spinal cord injury and its pathophysiology with the performance of subsequent clinical phases. The second part is dedicated to new treatment methods and approaches, with an emphasis on cell therapy. The third part presents treatment method using mesenchymal stem cells (MSCs), which immunomodulatory and anti-inflammatory properties, such as their ability to migration to the injured area and the possibility of genetic modification, made them a great candidates for the treatment of serious injuries. Keywords Spinal cord injury, stem cells, mesenchymal stem cells, cell therapy, regenerative medicine
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Mesenchymal stem cells and the possibility of their transdifferentiation into insulin producing cells.
Dostálová, Veronika ; Holáň, Vladimír (advisor) ; Čečrdlová, Eva (referee)
Mesenchymal stem cells (MSCs) have been demonstrated in almost all tissues of the body. Their main source is bone marrow and adipose tissue. These cells are multipotent, e.g. they are capable of differentiating into a variety of cell types. They are able to migrate into damaged tissues. Their other relevant property is a specific suppression of imunity. In the body they serve as precursors for specialized cell types and they also participate in formation of specific tissue microenvironment. Their properties represent a great potential in a wide range of clinical therapies. Besides other possible applications they could be used in the therapy of diabetes mellitus type one. During this disease insulin producing -cells are destroyed. MSCs have been used in experimental in vitro and in vivo studies to differentiate into insulin producing cells. However these cells are not able to produce sufficient amounts of insulin to exclude the supportive administration of exogenous insulin. Therefore there is a need for further research in this field of possible therapy.
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