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Electrospinning of Modified Biopolymers for Medical Applications
Pavliňáková, Veronika ; Martinová,, Lenka (oponent) ; Zajíčková, Lenka (oponent) ; Vojtová, Lucy (vedoucí práce)
Proposed dissertation thesis is dedicated to the preparation and characterization of novel biocompatible nanofibers with both biological and medical potential applications. The main emphasis of this thesis was focused on the preparation of composite nanofibers respecting the principles of "green chemistry", meaning hard requirements of tissue engineering. The theoretical part summarizes knowledge about the electrospinning process and its parameters. The literature review also describes the electrospinning of proteins like collagen and gelatin, their blends with synthetic polymers and biopolymers as well as with inorganic fillers. One chapter deals with various kinds of crosslinking agents to improve nanofiber hydrolytic stability. The last chapter is aimed to halloysite inorganic nanotubes (HNT) gaining much attention for the use as drug carrier due to its remarkable physical (mechanical reinforcement) and biological (biocompatibility and low toxicity) properties. The experimental part is divided into two chapters, each of them examines issues of nanofibrous material preparation from different perspective. The first part is focused on novel hydrolytically stable antibacterial gelatin nanofibers modified with oxidized cellulose. The unique inhibitory effect of the nanofibers was examined by luminometric method using genetically modified Escherichia coli strain. Seeded adenocarcinoma lung cells proved good adhesion and proliferation. Second experimental part explores the effect of source and amount of natural tubular halloysite on the structure and properties of biocompatible amphiphilic nanofibers based on a polycaprolactone and gelatin. The addition of HNT improved the thermal stability, mechanical properties (both stiffness and elongation) and reduced the crystallinity of nanofibers. The HNT from different sources did not affect the cell behavior but slightly influenced the proliferation and viability of cells on nanofibers.
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Polykaprolakton, jeho syntéza, charakterizace a degradabilita
Boháčová, Zdeňka ; Vojtová, Lucy (oponent) ; Hermanová, Soňa (vedoucí práce)
Předložená diplomová práce se zabývá studiem polymerace za otevření kruhu (ROP) kaprolaktonu katalyzované pomocí nových organických a organokovových sloučenin. V teoretické části práce jsou na základě literární rešerše popsány přehledy syntetických strategií a katalytických/iniciačních systémů pro ROP laktonů. V experimentální části byla provedena série polymerací -kaprolaktonu zaměřená na studium polymeračních podmínek (poměry rozpouštědlo/monomer, katalyzátor/iniciátor, monomer/iniciátor a koncentrace monomeru) v rozmezí teplot 25-70 °C. Provedená experimentální studie byla zaměřena na katalytické prekurzory na bázi organických karbenů (tBuNCH=CHN+tBu)CH Cl- (NHC-tBu) v roztoku tetrahydrofuranu a triazolového komplexu hliníku {O,O’-[4,5-P(O)Ph2tz]-AlMe2}, Ph = fenyl, tz = triazol, (OAlMe2) v roztoku chlorbenzenu. Připravené polymery byly charakterizovány pomocí 1H NMR spektroskopie (Bruker Avance), diferenciální skenovací kalorimetrie (TA Instruments Q 2000) a gelové permeační chromatografie (Agilent Technologies 1100 series). U připraveného vzorku s Mn = 12 kg/mol, Mw/Mn = 2,5 a se stupněm krystalinity 53 % byla otestována degradabilita účinkem mikroorganismu. Polymer ve formě lisované fólie a prášku byl vystaven působení bakteriálního kmene Bacillus subtilis (BS) v minerálním a živném médiu po dobu 42 dnů. Rastrovací elektronová mikroskopie (SEM) a konfokální laserová rastrovací mikroskopie (CLSM) potvrdily vývoj trhlin na povrchu filmu v porovnání s nezměněným povrchem kontrolních vzorků jako následek mikrobiálního ataku. Dále bylo pozorováno rozvinutí růžového zbarvení suspenze polymeru v důsledku aktivity mikroorganismu.
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Novel "green" catalysts for controlled ring-opening polymerization of lactide
Surman, František ; Vojtová, Lucy (oponent) ; Hermanová, Soňa (vedoucí práce)
The synthesis of polylactide (PLA) by ring-opening polymerization (ROP) of cyclic monomer can be realized by different routes. More than 100 catalysts for the synthesis of polylactide and other biodegradable aliphatic polyesters are published in the literature. For example organometallic catalysts based on Sn, Zn, Al etc. after finishing polymerization function became contaminants and using obtained polymer material in human body is controversial. At present, the research is focused on novel N-hererocyclic carbene catalysts. These metal-free catalysts are able to produce polymers with controlled molecular weight, narrow polydispersity, end-group fidelity with high reproducibility as well as to synthesize the block copolymers and complex macromolecular architectures, which is characteristic for living polymerization system. This diploma thesis is focused on study of polymerization of cyclic monomer D,L-lactide catalyzed by N-hererocyclic carbene. Polymerizations were carried out at the presence of benzylalcohol as initiator at THF. We were focused on the influence of composition of reaction system monomer – initiator – catalyst. Polymers of optically pure L-lactide with macroinitiators PEG with Mn of 1000 a 2000 g/mol were prepared as well. Number average molecular weight (Mn) and polydispersity index (PDI) was determined by GPC. 1H NMR was used to prove end-group fidelity.
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Determination of collagen degradation degree using spectroscopy
Fišerová, Kateřina ; Márová, Ivana (oponent) ; Vojtová, Lucy (vedoucí práce)
Presented diploma thesis deals with the characterization of collagen properties after exposure to various physical and chemical conditions. The main aim was to describe changes in the structure of collagen due to its modification, to assess the impact of these changes on the further use of collagen and the selection of appropriate methods for determining the collagen properties. Chemical structure of collagen, its use as biomaterials, collagen stability, and ways to determine the degree of disruption of the native collagen structure are described in the theoretical part. In the experimental work, water solutions based on the bovine collagen type I were treated by varying intensities of collagen disintegration (from 1 min at 6 000 rmp to 5 min at 14 000 rpm), different preparation temperatures (4 °C, room. temp. and 30 °C), preparation time (from 1 day up to 5 days), time of light radiation effect (from 1 day up to 14 days) and different values of pH (from 4 to 8). In order to describe the collagen structure and the changes that occurred during the modification of collagen by various conditions, prepared samples were analyzed both by UV VIS and FT-IR spectroscopy. Aqueous solution of 2,4,6 trinitrobenzensulfonic acid and ninhydrin solution in alcohol were used as the reagents with free amino groups of collagen in order to analyze the suitable colored products by UV-VIS spectroscopy. As for FT-IR spectroscopy, collagen samples were measured either in the form of KBr tablets or in films by ATR technique. To evaluate changes in the collagen structure the deconvulation of the amide I group characteristic band between 1590 - 1720 cm-1 in the infrared spectrum of collagen was used. The samples morphology was observed on the collagen lyophilized sponges by scanning electron microscopy (SEM). In accordance with theoretical assumptions, results confirmed that the largest disruption in collagen structure arise from long standing of collagen solution on the light. Significant changes cause as well increasing temperature and almost no effect on the collagen structure had disintegration intensity of collagen solution during the samples preparation. As for increasing pH, samples were stable and homogeneous until ph 5 but since the collagen has been separated and precipitated from water, which affected and distorted the UV-VIS analyses. The samples freezing freshly after preparation, disintegrated by 4 min at 14 000 rpm, at 30 °C and without changing the pH achieve the best characteristics. It was found that an appropriate method for determining the degree of collagen structure disruption is UV-VIS spectroscopy using 2,4,6 trinitrobenzensulfonic acid as reagent in combination with ATR infrared spectroscopy of collagen films.
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Biodegradable polyurethanes based on poly(ethylene glycol)
Kupka, Vojtěch ; Žídek, Jan (oponent) ; Vojtová, Lucy (vedoucí práce)
Presented diploma thesis deals with synthesis of biodegradable polyurethanes (bio-PUs) based on poly(ethylene glycol) (PEG) and poly(e-caprolactone) (PCL) with potential use in medicine. The aim of the work was to develop the methodology for synthesis of elastomeric polyurethane that could be formed into scaffold applicable in tissue engineering for regenerative human medicine. The theoretical part summarizes the knowledge of various kinds of materials which are possible to use for intended application. Scaffold forming techniques, their biocompatibility and characterization of properties of resulting materials are involved. The experimental part is focused on the development of suitable methodology to prepare functional samples based on PEG, PCL, hexamethylene diisocyanate (HMDI) and stannous octoate as the catalyst. Effect of bio-PUs composition (mainly different amount and molecular weight of PEG) on the swelling and hydrolytic stability was investigated together with testing mechanical properties, monitoring the net formation and the degree of conversion. The morphology of prepared samples was analyzed by optical microscopy, chemical composition was confirmed by infrared spectroscopy and the thermal properties were measured by differential scanning calorimetry. As for methodology development, it was found that it is necessary to degas all feedstocks before synthesis, otherwise non-well polymerized samples with different pore size have been obtained. Synthesis of bio-PU was realized under the nitrogen atmosphere in two steps. First, both polyols (PEG and PCL) were homogenized on the vacuum line at 130 °C followed by the addition of HMDI in the second step at 65 °C in glove-bag to obtain bio-PU samples. Finally samples were cured at the mould for 48 hours at 65 °C in oven. Based on the physical conditions of samples preparations, flexible bio-PU from white films through cloudy (with visible phase separation) to transparent films (clear) were obtained. Phase separation was confirmed by optical microscopy showing typical spherulite structure of crystalline phase (PCL) in amorphous matrix of PEG. The monitoring of net formation confirmed maximal conversion of polymerization (96 %) already after 4 hours of curing in the oven. Mechanical properties testing showed that important influence to rupture strength has isocyanate index (NCO/OH ratio). Different molecular weight of PEG showed influence to tensile properties as the specimens were completely cross-linked. Characterization of swelling exhibited increase the water uptake of samples with growing molecular weight of PEG (from 28 up to 58 wt.%). As well as, the bio- PU samples having higher molecular weight of PEG degraded faster in water at 37 °C. In presented diploma thesis was confirmed that it is possible to control the hydrolytic stability of obtained biodegradable polyurethane elastomers by the amount and the molecular weight of PEG.
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Poly(3-hydroxybutyrate) based materials for 3D printing in medical applications
Krobot, Štěpán ; Vojtová, Lucy (oponent) ; Přikryl, Radek (vedoucí práce)
This master's thesis deals with the preparation and testing of 3D printed scaffolds for bone tissue engineering. The aim of the thesis is laboratory preparation of polymer blends on the basis of poly(3-hydroxybutyrate), poly(lactic acid) and polycaprolactone and their processing into the form of 3D printing filaments. Three polymeric blends were prepared and processed into the form of 3D printing filaments. Differential scanning calorimetry was conducted to evaluate the thermal properties, followed by temperature tower test and warping test to determine the processing conditions for 3D printing. The lowest warping coefficient was 1.26 for a blend of poly(3-hydroxybutyrate) with polycaprolactone and plasticizer. Tensile test, three-point flexural test and compression test were used to study the mechanical properties of materials. Scaffolds with different surfaces for bone tissue engineering were 3D printed from prepared filaments to determine the most optimal surface for cell proliferation. To determine the surface properties and their influence on cell adhesion, optical contact angle measurement with the use of OWRK method to calculate surface energy was conducted. 3D printed surfaces were also subjected to roughness analysis by confocal microscopy to determine their roughness and its effect on contact angle with water and cell growth. Finally, in the last part, in vitro tests on scaffolds were conducted in collaboration with the Institute of Experimental Medicine (Czech Academy of Sciences) to find out whether the prepared materials are non-cytotoxic and how the surface of scaffold affects the cell growth and proliferation. In the end, two out of three materials were proven to be non-cytotoxic (both blends of poly(3-hydroxybutyrate) with polycaprolactone) and that their mechanical properties were comparable with human trabecular bone. The most optimal surface for cell growth is probably grid diameter 50 m with roughness along the perimeter 1.9 m, which corresponds with water contact angle 74.1°.
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Collagen cross-linking using oxidized cellulose
Filka, Pavel ; Márová, Ivana (oponent) ; Vojtová, Lucy (vedoucí práce)
Theoretical part of submitted diploma thesis deals with the basic finding of collagen, one of the most representing proteins in a human body, and oxidized cellulose used in medicine for several decades. Main part of the literature search is concerned on the collagen cross-linking, which is necessary for the collagen stability improving, thereby increasing the resistance against its degradation. As a cross-linking agent, the oxidized cellulose having the haemostatic properties as well as the carboxyl functional groups suitable for cross-linking proteins can be used. Experimental work was focused on the investigation of mutual behaviors of mixed water solutions based on oxidized cellulose and collagen. Films or lyophilized sponges prepared from these polymer mixtures could be used in medicine as a haemostatic or antibacterial wound healing coverings. The series of weight ratios between collagen and oxidized cellulose (9:1, 3:1, 5:3, 1:1, 1:2, 1:3, 1:9) involving constant content of collagen and increasing amounts of cellulose were prepared. Their ability to chemical linking, generating the formation of amide bond between carboxyl group of oxidized cellulose and free amino group of collagen, was investigated via two UV-VIS spectroscopic methods using a colored reaction among the chemical reagent (ninhydrin or 2,4,6-trinitrobenzenesulfonic acid) and free amino groups of collagen. Moreover, the carboxyl groups of oxidized cellulose were activated either in the polymer solution (in situ) or in the form of a film using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N hydroxysuccinimide (EDC/NHS). The changes at the secondary structure level were investigated by Fourier-transformed infrared spectroscopy (FT-IR). The stability of thin prepared films from each polymer mixture was determined using hydrolytic degradation at 37 °C. Morphological changes in the two types of samples, one frozen quickly at – 196 °C and second slowly at -30 °C, were observed using scanning electron microscopy (SEM). During the polymer mixture preparation, the precipitation up to the ratio of 1:1 between collagen and cellulose has occurred. UV-VIS analyses confirmed free amino groups reduction resulting in cross-linking between collagen and cellulose as well as increasing in hydrolytical stability obtained from degradation measurement of prepared films. At the ratio higher than 1:2 up to 1:9 the polymer mixture was homogeneous without precipitation, however, from the increasing free amino groups it is presupposed that cellulose acted as physical cross-linker at the small amount and beyond the equilibrium state works more like solvent. It probably caused the changes in collagen at the secondary structure level registered from FT IR analysis. Activation of cellulose carboxyl groups by EDC/NHS was not confirmed. Collagen to cellulose ratio affected as well as the porosity and the pore size of prepared scaffolds. Based on SEM, the porosity of scaffolds froze by liquid nitrogen was between 46 – 60 % up to the ratio of 1:1 and significantly increased with cellulose addition up to 81 % (at ratio of 1:9). Average pore size of neat collagen was very small (14 ± 5 m) in comparison with pure oxidized cellulose (79 ± 24 m). That is why the cellulose addition increased the pore size approximately up to 55 m except for the 1:9 ratio having very large pore size (186 ± 76 m) and very regular structure resembling honeycomb seen at pure cellulose as well.
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Effect of body fluids on setting, structure and mechanical properties of phosphate bone cements
Bednaříková, Vendula ; Michlovská, Lenka (oponent) ; Vojtová, Lucy (vedoucí práce)
Presented diploma thesis describes the preparation and characterization of composite calcium-phosphate bone cements (CPCs). In the literature review properties and structure of tricalcium phosphates (TCPs) are described, including their interaction with body environment. In the experimental work, first of all, sample preparation technique was determined by experiments with setting reactions provided in ultrapure water environment. Optimal technique of setting CPC samples included memory foam setting mold, ending setting reactions by absolute cold ethanol and vacuum drying procedures. Consequently, the work describes the sample preparation and process of TCP bone cement setting in both natural (pig blood) and simulated body fluids (physiological, Hank´s and Ringer´s solutions). Morphology study by Scanning Electron Microscopy (SEM) was performed for samples set for 1 day, 1 week, 2 weeks and 1 month, due to the significant change in crystalline structure proving as well by X-ray diffraction (XRD) analysis by determining -TCP conversion to calcium-deficient hydroxyapatite (CDHA). Porosity investigated by X-ray computed microtomography (-CT) was slightly higher at sample set in natural blood. Mechanical properties of CPC samples measured by mechanical compression tests showed stable cement strength set in physiological solution already after 1 day while cements set in blood has shown still increasing strength even at 1 month. On contrary, strength of cement samples rapidly decreased after 2 weeks of setting in both Hank´s and Ringer´s solutions probably due to its slightly acidic pH accelerating CPC disintegration. As a result, setting environment has significant effect on resulting CPC properties and natural blood in comparison to simulated plasma had shown better CPC properties while more closely imitating the in vivo conditions.
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Crosslinking of polysaccharide microfibers
Svidroňová, Barbora ; Abdellatif, Abdelmohsan (oponent) ; Vojtová, Lucy (vedoucí práce)
The general goal of the proposed diploma work was preparation and characterization of crosslinked hyaluronan microfibres, which would be stable in aqueous environment with proper mechanical properties. The fibres prepared by the wet spinning technique, were crosslinked with solution containing crosslinking reagent 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide or two crosslinking reagents, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, leading to amidation and formation of ester bonds. Characterization of prepared samples is the main goal of experimental part of the thesis. The swelling characteristics were carried out to determine the stability of fibres in three different solutions (with pH 7.4, 3 and 11). For determination of thermal stability was used thermogravimetrical analysis, and for determination of esterification and amidation was used infrared spectroscopy with Fourier transformation. Mechanical properties of fibres were studied by stress-strain tester. Additionally, the rheological properties were investigated, as well as the microstructure and surface of fibres by scanning electron microscopy. The fibres before chemical crosslinking had lower stability in all three types of solutions; the thermal stability was also lower, than the stability of crosslinked fibres. For not chemically modified fibres with crosslinking reagent, only one type of peak for esterification occurred. The infrared spectra of chemically crosslinked fibres showed the presence of two esterification peaks, which was the result of the effectiveness of the crosslinkig reagent. Amidation was also stronger for crosslinked fibres, especially for fibres crosslinked long time and in the solution with higher concentration of crosslinking reagent. Due to the heterogeneity of fibres, mechanical properties did not show evidence of any dependence on the crosslinking. The rheological study showed that the viscosity of fibre dissolved in water is less dependent on the shear rate than the powder of sodium hyaluronan dissolved in water. Despite more different methods of characterization of fibres which were used in this work, there are still many options for better characterization and closer understanding of this biopolymeric material.
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Novel organic materials for bioelectronic applications
Tumová, Šárka ; Vojtová, Lucy (oponent) ; Humpolíček,, Petr (oponent) ; Weiter, Martin (vedoucí práce)
This thesis is dedicated to material research focused on the field of organic bioelectronics. It aims to characterize and optimize novel materials for the future generation of bioelectronic transistor devices. Such devices are introduced first. Materials that are often used as their active layer and the challenges they bring and which this thesis addresses are mentioned. The attention is focused mainly on PEDOT:PSS, which is one of the most studied and most promising materials for bioelectronics. But despite all its advantageous properties, this material exhibits insufficient biocompatibility. This thesis discusses the possibilities of how to overcome such a drawback and shows approaches that have already been used for this. The specific PEDOT:PSS material modifications are then designed and implemented in the experimental part, aiming to provide a novel material suitable for bioelectronic applications that would represent an improved substitute for PEDOT:PSS, especially with regard to its biocompatibility. First, the modification of the PEDOT:PSS surface with the RGD peptide is shown, targeting the optimization of interactions of material with living cells. The immobilization of the peptide to the polymer surface was achieved using a photochemical approach and the sulfo-SANPAH molecule. The efficiency of such surface modification was studied using several methods, and its effect on the biocompatibility of the material was investigated by an MTT test. The experimental part further deals with the characterization and optimization of PEDOT:DBSA. To enhance its long-term stability, the cross-link using cross-linker DVS and GOPS was studied. The process involving molecule GOPS was investigated in detail, determining the optimal amount of such a dopant to obtain the stable PEDOT:DBSA thin film, its impact on the electrical properties of the resultant material, the effect of temperature on GOPS activity and the mechanism of cross-link. The electrical properties of studied materials were subsequently optimized using sulphuric acid post-treatment. The long-term stability of prepared films in an aqueous environment was examined by a delamination test, their biocompatibility was studied using the MTT test, and their electrical properties were investigated by the four-point probe method. To reveal the potential of proposed thin films for bioelectronic transistor applications, they were used as active layers of model devices and their performance was observed. The last chapter of the experimental part is devoted to the preparation of a conductive PEDOT:PSS hydrogel using DBSA molecule. The aim of this work was to prepare stable, easy-to-handle structures and to design a platform for their reliable electrical characterization. The rheology measurement was used to determine the optimal amount of DBSA leading to the formation of desired structure and to investigate the dependence of the mechanical properties of the hydrogel on its composition. Afterwards, the optimization of the platform for electrical measurement was conducted. The suitability of the interdigitated electrode system was studied together with the optimal electrode material. The platform was modified to prevent extensive evaporation of the dispersion medium from the hydrogel and to achieve system equilibrium. The appropriateness of the proposed platform was tested by long-term measurement of the I-V characteristics of the studied hydrogel.
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