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Study of Cellular Toxicity of Representative Nanoparticles in Tissue Cultures.
Filipová, Marcela ; Holada, Karel (advisor) ; Benson, Veronika (referee) ; Hubálek Kalbáčová, Marie (referee)
Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...
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Cell-free scaffolds functionalized with bioactive compounds in bone tissue engineering
Lukášová, Věra ; Rampichová, Michala (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Libusová, Lenka (referee)
There are good prospects for bone tissue engineering and therefore researcher is aimed towards the development of cell-free scaffolds. A cell-free scaffold serves as a temporal filling for critical size defects that do not heal spontaneously. Nevertheless, a suitable scaffold composition is yet to be discovered. Moreover, modifications of cell-free scaffolds with a drug delivery system activate the internal healing capacity. Platelets occur in the healing cascade as a natural source of growth factors (GFs), chemokines and cytokines. This autologous source of bioactive compounds enables the substitution of synthetic GFs. The aim of this thesis is to develop a bioactive cell-free scaffold with a drug delivery system supporting the physiological healing of bone defects. The centrifugal spinning method was used to produce nanofibrous poly-ε-caprolactone (PCL) scaffolds. PCL scaffolds were functionalized with different platelet concentrations. Bioactive compounds released from activated platelets were trapped within the formed fibrin net, enabling their gradual release. Improved metabolic activity, proliferation and alkaline phosphatase activity of MG-63 cells and human mesenchymal stem cells (hMSCs) were detected. The release of compounds lasted for two weeks and nearly reached the plateau phase,...
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The Cell-Nanomaterial Interactions and Their Application in Biomedicine
Vrabcová, Lucie ; Hubálek Kalbáčová, Marie (advisor) ; Pešta, Martin (referee) ; Benson, Veronika (referee)
Nanomaterials entered the biomedicine already at the beginning of the millennium and they still bring new and unique advances and possibilities for treatment, diagnosis and regeneration, thus they continue to facilitate the development of personalized medicine. Interactions at nanoscale allow until then unconceivable opportunities to influence processes on molecular level. The completely new perspectives of nanomaterial applications jointly raise attention concerning health and environmental safety issues. Although a lot of novel biomedical applications of nanomaterials have emerged, the vast majority is still found to be at the stage of a concept. The consistent basic in vitro research of elemental interactions of nanomaterial with biological environment should represent an essential part of its development. The concern of this thesis was to describe the cytocompatibility and interactions of two types of nanomaterials with different human cells. First, ultra-fine grain titanium was tested for prospective use in implant development. We confirmed its positive effect mainly on the growth of osteoblasts and recommended the further pre-clinical trials of this material in a form of a bone or dental implant. Second, several types of ultra-small (˂ 5 nm) nanoparticles of different origin (silicon, gold...
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L1CAM and its role in cellular senescence
Mrázková, Blanka ; Hodný, Zdeněk (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Rohlena, Jakub (referee)
Cellular senescence, originally defined as irreversible cell cycle arrest, was shown to act in organism as a double-edged sword. On the one hand, cellular senescence is considered an anti-cancer barrier and it beneficially contributes to processes such as wound healing and tissue regeneration. On the other hand, its longer persistence in the organism, usually when not eliminated by the immune system in elder age, cellular senescence contributes to age-related diseases and ageing itself. Moreover, senescent cells emerge as a result of radio- and chemo- therapy and can lead to detrimental effects when not eliminated. There is also accumulated evidence that senescent cells can overcome the proliferation barrier and become malignant (often after a cancer therapy) rendering senescence original definition invalid. In effort to improve the quality of health and life and to minimize the cancer risk after therapies, senescent cells have become one of the most interesting subjects for a targeted therapy. There is a promising potential in developing effective tools, namely drugs specifically killing senescent cells or reducing their detrimental effect (senolytics) that focus on senescent cells elimination in order to rejuvenate the organism, to extend a life span, and to prevent ageing-associated diseases and...
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Biocompatibility of diamond-based platforms with neuronal cells.
Gottfriedová, Kristýna ; Benson, Veronika (advisor) ; Hubálek Kalbáčová, Marie (referee)
5 Abstract Microelectrode arrays represent therapeutic approach to neurodegenerative diseases treatment. The development of electrode platforms is rather challenging due to the direct interaction of the material with neuronal tissue. Nanodiamond is one of the researched materials because of its biochemical properties: biocompatibility with many cell types, chemical inertness, high wear and corrosion resistance. During nanodiamond platforms development, biological research focuses on biocompatibility of used material with primary neurons and the evaluation of their adherence to the nanodiamond that is important for recording of electrical activity of neurons. Both, the biocompatibility as well as the adherence depend on the used nanodiamond, manufacturing, roughness, and surface modification. The amount of boron dopant enabling electric conductivity of nanodiamond is also important. In my thesis, I have summarized research on biocompatibility and adherence of neural cells on behalf of above mentioned parameters. I discuss here the variability of experimental results in order to surface modification and cultured cells type. According to the research reports, it seems that neuronal cells prosper well and prefer to adhere to platforms covered with molecules of extracellular matrix or at least poly-lysine...
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Biomolecular corona of Si and Au nanoparticles and its impact on interaction with cells
Javorová, Pavlína ; Hubálek Kalbáčová, Marie (advisor) ; Fišer, Radovan (referee)
Biological response to presence of gold and silica nanoparticles is extensively researched area of science. However there is only limited knowledge and understanding of the effects of small and ultrasmall nanoparticles. Regarding the unique physical and chemical properties that originate from the small size have these nanoparticles ability to interact very specifically on molecular level with organisms. Once the particle enters the complex physiological environment of the body molecules (predominantly of protein character) adsorb on the surface and form a polymeric case called biomolecular corona. There is a presumption that the first contact of the nanoparticle with the cell is mediated through the molecules of this corona and are important in subsequent steps of interactions of nanoparticle-biocorona complex. Therefore the genesis and structure of biocorona is as essential as the structre of the nanoparticle itself. Nanoparticles enter and are internalized within the cell and cellular compartments through the same mechanisms like naturally occurring molecules and substances. There are slightly different patterns of behavior of small and ultrasmall nanoparticles that are not fully researched and understood. Response of mamallian cells to the presence of the ultrasmall nanoparticles is very...
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Biocompatibility of diamond-based platforms with neuronal cells.
Gottfriedová, Kristýna ; Benson, Veronika (advisor) ; Hubálek Kalbáčová, Marie (referee)
Existuje mnoho typů platforem, které jako povrch, bezprostředně interagující s buňkami, využívají diamant. Hlavní rozdíly jsou v typu použitých diamantů. Jedná se o nanodiamanty a ultrananokrystalické diamanty, které mohou být různě strukturované a také dopované o atomy boru. Ačkoli by pravděpodobně bylo hezké, kdyby na platformách nemusela být žádná povrchová úprava. Podle výsledků výzkumů to nevypadá jako lehký úkol, výzkum v tomto směru probíhá, ale zatím bez významných úspěchů. Nejlepších výsledků se při kultivaci buněk na platformách dosahuje, když je povrch potažen alespoň částečkami molekul extracelulární matrix. Jako o trošku méně účinné se jeví využití molekul, které na základě elektrostatických sil navodí adhezi buňky. Jedná se například o molekulu lysinu. Dalším krokem ve výzkumu je ověření, zda povrchy, které se v první fázi zkoumání jevily jako biokompatibilní, budou i po dopování stále optimální pro růst buněk. Z níže uvedených výzkumů zatím vyplývá, že například dopování nanodiamantu borem nemá významný vliv na prosperitu buněk. Klíčová slova nanodiamant, nervová buňka, biokompatibilita, mikroelektrodové pole
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Studium interakce buněk s biomimetickým materiálem a jeho využití v biomedicíně
Sauerová, Pavla ; Hubálek Kalbáčová, Marie (advisor) ; Vandrovcová, Marta (referee) ; Jendelová, Pavla (referee)
Biomaterials are considered as very promising tools for regenerative medicine. They have compensatory or supporting function in organism and they are often developed to support specific conventional medical procedures. So-called biomimetic materials are developed to imitate natural environment of organism and to induce positive innate responses of organism. An essential part of biomaterial development is in vitro biological evaluation, which characterizes (often for the first time) the potential of developed material for its clinical application. This Ph.D. thesis deals with in vitro biological evaluation of three different biomimetic materials. In all three cases, the comprehensive evaluation was an integral part of the material development and optimization processes. Each material was in vitro characterised at the level of cell-material interactions with respect to its intended specific application.. In the first part, cell response to potential drug delivery system based on colloidal complexes of cationic surfactants with hyaluronic acid (HyA) was characterized. HyA protection ability and its limits were described; also the role of fetal bovine serum (FBS) in cell response to the stress stimuli was confirmed. Results considered surfactant-HyA complexes as promising system for drug delivery. In...
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Studies of properties of viral capsid proteins and development of recombinant vaccines and diagnostic components based on artificial viral structures
Fraiberk, Martin ; Forstová, Jitka (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Hejnar, Jiří (referee)
The aim of this study was to develop a system for easy production of different veterinary chimeric vaccines based on stable mouse polyomavirus (MPyV) structures. The system is designed for antigens that are problematic in production or stability. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers based on the major capsid protein VP1 were designed to be exploited as vaccines against other pathogens. The different strategies used in this study are based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by inserting them into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, thus forming giant pentamers of a chimeric protein. Candidate vaccine antigens against porcine circovirus 2 (PCV2), the causative agent of porcine circovirus 2 systemic diseases (PCV2-SD) which causes significant economic losses in swine breeding, were prepared using the constructed vectors. All candidate vaccines induced the production of antibodies against the capsid protein of PCV2 after immunization of mice. The candidate vaccine Var C based on fusion of MPyV and PCV2 capsid proteins, is able to induce production of antibodies with...
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