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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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Carbon nanoparticles as promising components of materials for bone tissue engineering
Grausová, Ľubica ; Bačáková, Lucie (advisor) ; Mikšík, Ivan (referee) ; Balík, Karel (referee)
Recently, nanotechnology in tissue engineering has become a very irnportant field of study. Many different materials of nanoscale roughness have been studied for their potential use in the regeneration of various tissues. The reason is that nallostructured materials imitate the architecture of natural extracellular matrix and thus support cell adhesion, growth and differentiation. Our investigations were focused on the influence of fullerene layers, carbon nanotube-terpolymer composites and nanocrystalline diamond layers on the adhesion. growth and differentiation of human osteoblast-like MG 63 cells. Each of these materials supported colonization with cells. On continuous fullerene C6s layers, deposited on composites with the carbon matrir reinforced with carbon fibres, the cell population density was lower than on non-coated composites. but MG 63 cells were well-spread w'ith rvell-developed vinculin-containing focal adhesiort plaques and a beta- actin cy'toskeleton. On contposites of carbon naliotubes u ith a terpolymer of poll.tetrafluoroethvlene. poly propy lene and polyvinyldifluoride, the adhesion, spreading. formation oť tbcal adhesion plaques and actin cytoskeleton, viability and cell grouth u ere markedly improved in comparison with pure terpolymer. At the same time, these cells did not...
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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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Growth of human adipose-derived stem cells on Ti-6Al-4V alloy with various surface modifications
Kročilová, Nikola ; Bačáková, Lucie ; Pařízek, Martin ; Havlíková, Jana ; Motarjemi, H. ; Molitor, M. ; Gabor, R. ; Marvan, J.
Adipose-derived stem cells (ASCs) are of a great importance for bone tissue engineering. We compared ASCs obtained by liposuction under two pressures (-200 mmHg and -700 mmHg). The number and proliferation activity of cells isolated under -700 mmHg (ASC-H) were higher than in cells isolated under -200 mmHg (ASC-L). However, the ASC-L was more active in osteogenic differentiation, as manifested by a higher intensity of fluorescence of alkaline phosphatase and osteocalcin in these cells. The adhesion and growth of ASCs were then studied on Ti-6Al-4V samples either unmodified (K, roughness parameter R-a = 280 nm) or modified by shot blasting and tarnishing A, R-a = 200 nm), vibratory finishing (B, R-a = 100 nm) and vibratory finishing, shot blasting and polishing (C, R-a = 80 nm). All modified samples were more wettable than the K samples. On day 1 after seeding, the size of cell spreading area on some modified samples was lower than on K samples, namely on B samples in ASC-L, on B and C in ASC-H and on A in control MG-63 cells. On day 3, the cell number on some modified samples became higher than on K samples, namely on C samples (ASC-L), on B (ASC-H) and on B and C s (MG-63 cells). On day 7, the numbers of ASC-H and MG-63 cells on all modified samples evened out and became significantly higher than on K samples. On ASC-L samples, the highest cell numbers were obtained on A samples. Thus, all studied modifications of Ti-6Al-4V enhanced growth of ASCs and human osteoblast-like MG-63 cells.
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Growth and differentiation of human osteoblast-like cells on TI-6AL-4V alloy modified with anodization
Doubková, Martina ; Bačáková, Lucie ; Pařízek, Martin ; Vandrovcová, Marta ; Musílková, Jana ; Lisá, Věra ; Gabor, R. ; Marvan, J.
Titanium and its alloys are widely used as hard tissue implants for their high biocompatibility and suitable physical properties. Along with other surface treatment methods, anodization technique is known to enhance osseointegration. The aim of this study is to evaluate the adhesion, growth and osteogenic differentiation of human osteoblast-like SAOS-2 cells on Ti-6Al-4V samples anodized in electrolytes composed of a stable volume of KOH (336.48 g/l) and variable volumes of liquid glass (124.47 g/l, 84,12 g/l and 37.38 g/l; samples s5, s6 and s7, respectively). Non-anodized Ti-6Al-4V samples, cell culture polystyrene (PS) and microscopic glass coverslips served as control materials. On days 2 and 4 after seeding, the cell number did not differ significantly among the tested samples. However, on day 7, the cell number on s6 samples reached the lowest values, which could be attributed to a non-homogeneous TiO2 film on s6 samples formed during anodization. Nevertheless, the osteogenic differentiation, estimated by the intensity of fluorescence of collagen I in cells grown in a differentiation medium, was the highest on s6 samples. On s5 samples, coated with homogeneous TiO2 films, both cell numbers and intensity of fluorescence of collagen I was relatively high. The bone matrix mineralization, evaluated by Alizarin Red staining, was the highest on s5 samples in standard culture medium, and similar on all tested samples in differentiation medium. Thus, the surface modification of s5 samples could be considered the most suitable for application in bone implants.
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Polymeric nanofibrous scaffolds reinforced with diamond and ceramic nanoparticles for bone tissue engineering
Bačáková, Lucie ; Pařízek, Martin ; Staňková, Ľubica ; Novotná, Katarína ; Douglas, T.E.L. ; Brady, M. A. ; Kromka, Alexander ; Potocký, Štěpán ; Stránská, D.
Three types of nanofibrous scaffolds were prepared by electrospining: (1) poly(lactide-co-glycoside) (PLGA) scaffolds reinforced with 23 wt.% of diamond nanoparticles (DNPs), (2) poly(L-lactide) (PLLA) scaffolds with DNPs in concentration ranging from from 0.4 wt.% to 12.3 wt.%, and (3) PLLA scaffolds with 5 wt.% or 15 wt.% of hydroxyapatite (HAp) nanoparticles. The diameter of the nanofibers ranged between 160 and 729 nm. The nanofibers with nanoparticles were thicker and the void spaces among them were smaller. Mechanical properties of the nanoparticle-loaded scaffolds were better, as demonstrated by a rupture test in scaffolds with DNPs and by a creep behavior test in scaffolds with HAp. On PLGA scaffolds with DNPs, the human osteoblast-like MG-63 cells adhered in similar numbers and grew with similar kinetics as on pure PLGA scaffolds. Human bone marrow mesenchymal stem cells grew faster and reached higher population densities on PLGA-DNP scaffolds. However, on PLLA-based scaffolds, the activity of mitochondrial enzymes and concentration of osteocalcin in MG-63 cells decreased with increasing DNP concentration. On the other hand, the metabolic activity of MG-63 cells and content of osteocalcin in these cells were positively correlated with the HAp concentration in PLLA scaffolds. Thus, PLGA nanofibers with 23 wt% of DNPs and PLLA nanofibers with 5 and particularly 15 wt.% of HAp seem to be promising for bone tissue engineering.
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