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Biomedical applications of cationic gold nanoparticles
Žárská, Monika ; Hodný, Zdeněk (advisor) ; Králová, Jarmila (referee) ; Bačáková, Lucie (referee)
Cationic gold nanoparticles (GNPs) represent innovative materials promising for treating severe diseases, including cancer. Due to the unique physical properties of colloidal gold, GNPs have been shown to function as theranostics agents, allowing the diagnosis and treatment of the pathological area simulataneously. In addition, a cationic surface charge of GNPs provides extensive nanoparticle-cell interactions. However, despite the great potential in clinical medicine, various types of GNPs have contradictory results, and the studies dealing with the biological and therapeutic properties of cationic GNPs are inconsistent. This doctoral thesis summarizes the current state of knowledge about the biological properties and medical applications of GNPs focusing specifically on positively charged GNPs. A overview of plasmonic photothermal therapy (PPTT) as a cancer treatment strategy is included. Using a step-by-step research approach, our results then characterize the therapeutic potential of GNPs with a specific shape of nanorods (GNRs) and surface modification with quaternary ammonium salt (QAS). At first, the elementary factors participating in the interaction of cationic GNRs with cells, their transmembrane and intracellular transport, and long-term persistence in cells were described. Subsequently,...
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The co-culture of keratinocytes and fibroblasts on a multi-layered polyester nanofibrous membrane enriched with platelet lysate
Blanquer, Andreu ; Filová, Elena ; Jenčová, V. ; Brož, Antonín ; Kuželová Košťáková, E. ; Lisnenko, M. ; Procházková, R. ; Bačáková, Lucie
The prevalence of chronic wounds is increasing due to the population ageing and specific illnesses like diabetes mellitus and vascular diseases. Nanofibrous membranes fabricated using synthetic polymers are promising materials to enhance skin wound healing. PCL and PVA membranes are being studied to be used as scaffolds for skin tissue engineering and hydrogels for controlled drug delivery, respectively. The present study considers the development of a multi-layered membrane made of PCL and PVA loaded with platelet lysate (PL). PCL nanofibers allowed cell adhesion and growth, whereas PVA acted as a hydrogel that releases the bioactive compounds of platelet lysate. The cytocompatibility of the membranes containing PL and without it was demonstrated on two cell types involved in wound healing, i.e. keratinocytes and fibroblasts. Both cell types were able to adhere and proliferate on the membranes. In addition, the membrane containing PL enhanced the proliferation of fibroblasts. A co-culture study was also performed by seeding each cell type on one side of the membrane. The cells were co-cultured for 7 days and the results showed that PL increased the proliferation of cells achieving a monolayer of keratinocytes or fibroblasts on each side of the membrane. Thus, the beneficial effect of PCL-PVA+PL membranes on monocultures and co-cultures of skin cells was demonstrated, and these membranes can be considered potential scaffolds for treatment of chronic wounds.
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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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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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Vascular and osseous cells in polymer structures for tissue engineering
Filová, Elena ; Bačáková, Lucie (advisor) ; Maxová, Hana (referee) ; Motlík, Jan (referee) ; Kromka, Alexander (referee)
Artificial vascular and bone prostheses are engineered as bioinert, not allowing cell attachment and growth. Our aim was to prepare materials based on natural and synthetic polymers that could modify the surface or create the bulk material of prostheses, and test their bioactivity in vitro. We prepared fibrin assemblies of various thicknesses and evaluated the adhesion, growth and differentiation of endothelial cells (EC) on these layers. We observed increased cell spreading on twodimensional fibrin assemblies and improved cell growth and maturation on thick fibrin gels. Fibrin coated with collagen I, or fibronectin, increased the adhesion area and the proliferation activity of vascular smooth muscle cells (VSMC). Synthetic polymers were based on an inert block copolymer of poly(DL-lactide) and polyethylene oxide (PDLLA-b-PEO) in which 5% or 20% of the PEO chains were grafted with Gly-Arg-Gly-Asp-Ser-Gly oligopeptide, a ligand for cell adhesion receptors. Grafting oligopeptide peptide to the cell non-adhesive copolymer restored adhesion and growth of VSMC, even in a serum-free medium. Synthetic polymers could therefore serve as artificial extracellular matrix analogues for vascular tissue repair and regeneration. Our study with human osteoblast-like MG 63 cells cultured in poly(lactic-co-glycolic acid)...
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Nanofibrous materials in bone tissue engineering
Zajdlová, Martina ; Bačáková, Lucie (advisor) ; Míčová, Petra (referee)
This thesis focuses on nanofibrous materials which are highly promising regarding they application in a modern interdisciplinary scientific field - tissue engineering. Through the years there have been developed various strategies for creating materials usable in tissue engineering. The earliest materials that were made did not allow any cell adhesion on their surfaces (so-called "bioinert" materials), whereas nowadays there is an effort to create hybrid bioartificial organs. Especially in bone tissue engineering do polymeric materials in the form of a nanofibrous network, such as polylactide or polycaprolactone with the addition of inorganic particles (for example nanocrystalic hydroxyapatite), show great potential. Such materials mimic the natural bone tissue and stimulate the adhesion, proliferation and differentiation of cells into desirable a cell type. In the experimental part of this thesis one of these promising nanomaterials was tested for its biocompatibility in vitro. Polylactide in the form of nanofibrous networks with 0, 5 and 15 % of nanocrystallic hydroxyapatite was provided by Elmarco s.r.o, Liberec. Human osteoblast-like cells MG 63 were cultivated on these materials for 1, 3 and 7 days. The results show the convenience of hydroxyapatite particles which stimulate the cells to the...
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Interactions of vascular and bone cells with bioactive polymers for construction of tissue replacements
Pařízek, Martin ; Bačáková, Lucie (advisor) ; Smetana, Karel (referee) ; Hrubý, Martin (referee)
This work deals with the interaction of cells with surface-modified existing or newly created materials developed for vascular and bone tissue engineering, and also for controlled drug delivery into implants. In the first part of this work, we modified the surface of the polyethylene foil by Ar plasma, and then we grafted them with bioactive molecules (glycine, polyethylene glycol, albumin) and with C or Au nanoparticles. These modifications improved the chemical and physical characteristics of the material for the adhesion and growth of vascular smooth muscle cells (VSMC), and also for their phenotypic maturation towards the contractile fenotype. In future, these modifications can be also used for material currently used for fabrication of clinically used vascular prostheses in order to increase their biocompatibility. The aim of the second part of this work was to develop a perivascular drug-delivery system that would release the antiproliferative drug Sirolimus. This perivascular system is designed to be wrapped around a venous graft, implanted to the arterial position, such as in the case of the aortocoronary bypass. The system comprises a polyester mesh, which ensures the mechanical stability of the system and of the venous wall, and a copolymer of L-lactide and ε-caprolactone (Purasorb), serving as a...
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Interaction of bone and vascular cells with materials constructed for tissue replacement and drug delivery system
Vandrovcová, Marta ; Bačáková, Lucie (advisor) ; Smetana, Karel (referee) ; Jirka, Ivan (referee)
Interactions between biomaterials and cells play an important role in tissue engineering. It has been repeatedly shown that the cell behaviour strongly depends on physical and chemical properties of the material surface. In our study we focused on materials used for the construction of bone implants and replacements to support cell adhesion, growth and osteogenic diferenciation and to lead to an integration between an implant and a bone tissue. First we tested an influence of different micrpattern of fullerens' C60 and composites' C60/Ti films to adhesion of bone cells MG 63, their initial spreading, growth, viability and formation of cytosceletal protein actin. Some of these films were additionally iradiated with Au+ ions, which led to the conversion of some C60 molcules into amorphous carbon (a-C). We confirmed that pattern influenced the distribution of cells without decrease viability. Thus, the production of patterns could be used to direct cell adhesion. In the second study, we also observed the influence of the thickness of coated layer. We found that more selective cell growth can be achieved by depositing of thicker fullerens' film. Our study with osteoblast-like MG 63 cells cultured on PLGA films revealed, that surface coated with chosen components of extracellular matrix (colagen I and...
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The biocompatibility and potential cytotoxicity of materials for joint replacement manufacturing and coating
Kopová, Ivana ; Bačáková, Lucie (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Jendelová, Pavla (referee)
Currently used prostheses for total joint replacement still have numerous disadvantages: extreme stiffness or elastic modulus of the bulk metallic material; insufficient integration of the implant into the host bone; and a high wear and corrosion rate, which causes an accumulation of mostly metallic or polymeric wear debris. Because of these reasons, many patients experience increasing local pain, swelling, allergic reactions, and inflammation resulting in bone loss and the aseptic loosening of the implant leading to the need for painful and expensive revision surgery. To address the mechanical issues of commonly used orthopaedic alloys, this thesis presents the development of the new β-type titanium alloy Ti-35Nb-7Zr-6Ta-2Fe-0.5Si with a relatively low elastic modulus (up to 85 GPa), increased tensile strength (880 MPa), and enhanced biocompatibility and osteoconductivity. Considering the generally low osteoinductivity of metallic implants, various surface modifications and coatings have been developed to improve the cell-material interaction, e.g. carbon-based coatings. Among these coatings, C60 fullerene layers have emerged as a great candidate for coating orthopaedic implants due to their therapeutic potential in arthritis. The potential cytotoxicity and DNA damage response of fullerenes have...
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