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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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Synthesis and Characterization of Multifunctionalized Biodegradable Copolymers
Michlovská, Lenka ; Petrůj, Jaroslav (oponent) ; Lehocký,, Marián (oponent) ; Vojtová, Lucy (vedoucí práce)
This dissertation summarizes the current state-of-the-art in the field of biodegradable thermosensitive copolymers, which in the form of aqueous sol at room temperature change to gel at body temperature. These polymer materials are useful in medicine for injection application as drug carriers or resorbable implants for tissue regeneration. In experimental work, thermosensitive amphiphilic triblock copolymers based on hydrophobic biodegradable polylactide and polyglycolide and biocompatible hydrophilic polyethylene glycol (PLGA–PEG–PLGA) were synthesized using vacuum line apparatus. Prepared PLGA–PEG–PLGA copolymer with two phase transitions from sol-gel and gel-suspension was subsequently modified with itaconic anhydride. The resulting functionalized ITA/PLGA–PEG–PLGA/ITA copolymer contains at both ends of chains reactive double bonds suitable for further polymerization or crosslinking and functional carboxyl group for possible modification by biological active substances. Physical and chemical crosslinking were also investigated in terms of the ratio of hydrophilic and hydrophobic chains and with a view of the amount of bounded itaconic acid. Prepared aqueous solutions of synthesized ITA/PLGA–PEG–PLGA/ITA copolymer change to a gel at the temperature between 33 and 43 °C. The evaluated critical gel concentration and the critical gel temperature was 6 % w/v and 34 °C, respectively, for the copolymer with PLGA/PEG ratio equal to 2.5. When the copolymer was more hydrophobic, then start of gelation became earlier and gel was more hydrolytically stable. Gel stiffness increased with increasing PLGA/PEG ratio and it depends on methods and type of solvent used during purification of copolymer. Prepared ITA/PLGA–PEG–PLGA/ITA copolymers were crosslinked using blue light without further crosslinker. Hydrolytical stability of ITA modified samples was significantly improved and increased in direct proportion with the both increasing time of crosslinking and the amount of double bonds attached to polymer chains. Sample having 63 % of ITA crosslinked for 40 mins fully degraded in water after 32 days. By proton NMR relaxometry it was found that while the sample has been swelled in water (after 12 hours) the amount of unbonded water reduces and gradually diffuses into cavities on the surface of sample and slowly changed to both weakly bonded water and strongly bonded water to polymer chains. Weakly bonded water began release from sample and changed back to the free water, when sample start to degrade and nodes and gel network begin to break. However, the thermal stability of chemically crosslinked samples increased only up to 20 minutes of crosslinking time where approximately 57 % of double bonds of itaconic acid (at 1640 cm-1) were transformed to the new single RR'C–CHR'' bonds at 795 cm-1ones by making crosslinks proved by ATR-FTIR. Longer crosslinking time (above 30 minutes) led to changes in chemical structure by beta-scission of chain and partially by recombination of double bonds. Rediscovery of new double bonds in different place of the chain reduced both the thermal stability and glass transition temperature from 242 °C to 237 °C and from -2.2 to -5.8 °C, respectively. The proposed thesis shows how the polymer composition, modification by functional groups and physical conditions affect either the physical or the chemical crosslinking of prepared amphiphilic copolymers.
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Functionalization of biodegradable polymers by itaconic anhydride
Michlovská, Lenka ; Hermanová, Soňa (oponent) ; Vojtová, Lucy (vedoucí práce)
Presented diploma thesis describes preparation of biodegradable termosensitive triblock copolymer based on poly(ethylene glycol), poly(lactic acid) and poly(glycolic acid) (PLGA-PEG-PLGA) that was subsequently modified by itaconic anhydride (ITA), which gives copolymer both reactive double bonds and functional carboxylic acid groups essential for the reaction with biological active material. The general goal was optimizing reaction conditions in order to reach the highest yield of ITA end-capped to polymer resulting in ITA/PLGA-PEG-PLGA/ITA copolymer. Prepared functionalized copolymer as a component of heterogeneous composite e.g. with hydroxyapatite might be suitable for biomedical application in the field of tissue engineering as a temporary replacement or adhesive of hard tissues (bones). In the theoretical part, hydrogels, their separation, crosslinking and degradation mechanism are generally described together with physico-chemical properties and the synthesis of the individual used biomaterials and their copolymers, itaconic anhydride and its functionalization. The experimental part describes in detail the synthesis of PLGA-PEG-PLGA copolymer via ring opening polymerization (ROP) using vacuum line and Schlenk’s techniques. Kinetics of the ROP was measured and optimization of polymerization conditions was suggested. Prepared thermosensitive copolymer was additionally modified by itaconic anhydride via catalytic ring-opening reaction. Optimization of ITA functionalization conditions were evaluated in terms of effect temperature, time, ITA purification and presence of the solvent effect. Successful end-capping of PLGA-PEG-PLGA copolymer by ITA was precisely characterized by means of 1H NMR, FT-IR and GPC methods. Kinetics of PLGA-PEG-PLGA copolymerization from unsublimated (neat) and sublimated (purified) D,L-lactide and glycolide were studied. In both cases the synthesis proceeded in a bulk at 130 °C for 3 hours with conversion approximately of 90 %. Prolonged polymerization period had no effect on the increase of conversion. In the case of ROP using unsublimated monomers, a rapid increase of monomer conversion was observed during first few minutes, followed by constant progress. Resulting copolymer displayed molecular weight of 7155 g/mol and narrow polydispersity index of 1.26. Optimal conditions were reached when sublimated monomers were polymerized. First, increase of conversion up to 88 % was nearly linear (living polymerization) to 2.5 hours, after that a plateau was observed. Well-defined PLGA-PEG-PLGA copolymer with molecular weight of 7198 g/mol and narrow polydispersity index of 1.20 was obtained. Optimal conditions for synthesis of ITA/PLGA-PEG-PLGA/ITA copolymer were reached with sublimated itaconic anhydride in a bulk at the temperature of 110 °C with total reaction time of 1.5 hours. As a result 76.6 mol. % of ITA was end-capped to the original PLGA PEG PLGA copolymer. Resulting molecular weight of ITA/PLGA-PEG-PLGA/ITA copolymer (5881 g/mol) with polydispersity index of 1.37 found by GPC correlated well with Mn calculated from 1H NMR and a theoretical Mn (Mn(theor)/Mn(GPC)/Mn(NMR) = 1/0.89/0.96).
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Ocenění podnikatelského subjektu
Opluštilová, Lucie ; Michlovská, Lenka (oponent) ; Zinecker, Marek (vedoucí práce)
Cílem předkládané diplomové práce je stanovit subjektivní hodnotu akciové společnosti VINSELEKT MICHLOVSKÝ za pomocí vybraných výnosových metod oceňování. Ocenění bude provedeno k 1. 1. 2011 a bude sloužit pro interní potřeby managementu. Teoretická část objasňuje hlavní pojmy a charakterizuje metodologické po-stupy, které souvisí s oceňováním podniku. Teoretická východiska literární rešerše jsou rozpracována a aplikována na konkrétní podnikatelský subjekt v další části. Praktická část představuje oceňovanou společnost, zahrnuje výsledky strategické a finanční analýzy a v konečné fázi i návrh ocenění.
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Crosslinking of thermosensitive functionalized copolymers by blue light
Křivánková, Nikola ; Michlovská, Lenka (oponent) ; Vojtová, Lucy (vedoucí práce)
The aim of this thesis was to prepare a hydrogel with a hybrid network of only one type of biodegradable copolymer. The new degradable hydrogel, containing both physical interactions (arising at physiological temperature of 37 °C) and chemical bonds initiated by blue light could be used as a resorbable wound dressing or as an injectable carrier with a gradual and well controlled drug release. Thermosensitive PLGA–PEG–PLGA copolymer synthesized by living ring-opening polymerization was subsequently functionalized with itaconic anhydride to form ITA/PLGA–PEG–PLGA/ITA light-sensitive and temperature-sensitive macromonomer. At 37 °C, the copolymer forms a micellar network due to hydrophobic interactions. Itaconic acid double bonds, which are attached to the ends of the copolymer chain, allow photochemical crosslinking of micelles with a view to increase the hydrolytic stability of novel hydrogel. The synthesized copolymers were characterized by GPC and 1H NMR methods. The formation of a physical network at physiological temperature was confirmed by rheological analysis. The physically crosslinked ITA/PLGA–PEG–PLGA/ITA hydrogel was then irradiated with blue light (430 – 490 nm) in the presence of a water soluble biocompatible photoinitiator LiTPO and chemically characterized by ATR-FTIR. The resulting hydrogel was transparent, flexible, absorbed up to 1176 % water, and was stable for 20 days in saline at 37 °C. The ITA/PLGA–PEG–PLGA/ITA hydrogel with hybrid network was also prepared in the presence of a crosslinker PEGDA, that significantly reduced the time required for crosslinking the hydrogel, but further analyses are needed to more fully understand the principles of the novel hydrogel types.
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Rheological properties of modified polymer-composite bone pastes
Hlináková, Kristýna ; Vojtová, Lucy (oponent) ; Michlovská, Lenka (vedoucí práce)
Diploma thesis is focused on study of visco-elastic behavior of bone pastes based on calcium phosphate and the aqeous solution of biodegradable termosensitive triblock copolymer improving flowing properties of the paste. In the theoretical part, a brief characterization of calcium phosphate cements is elaborated. It also deals with the characteristics of rheological properties of injectable bone pastes. It includes a brief overview of additives influencing the rheological and mechanical properties of pastes. The experimental part was firstly focused on the triblock copolymer characterization by proton nuclear magnetic resonance spectroscopy and rheology. Secondly, the preparation of modified polymer-phosphate pastes was evaluated in terms of visco-elastic properties. Bone paste was modified by the addition of adhesive compouds (dopamine and sodium iodate) and antibacterial compound (selenium nanoparticles). Visco-elastic properties were performed by rheological analysis, during which the setting process proceeded exhibiting significant changes in the thixotropic behavior of both unmodified and modified phosphate pastes. The setting process took place at 23 °C and 37 °C imiting physiological environment. Morphology of polymer-phosphate pastes was characterized by scanning electron microscopy and the particle size was determined using a laser particle analyzer. It has been shown that the above-mentioned additives had a positive effect on the kinetics of self-setting paste process, moreover, selenium nanoparticles improved as well thixotropic behaviors of polymer-phosphate pastes. Therefore, novel injectable composite bone pastes are suitable for mini-invasive surgery in terms of treating fractures due to the adhesion additives as well as for osteomyelitis treatment due to the possible release of antibacterial nanoparticles.
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Modification of thermosensitive copolymer with bioactive substances for medical applications
Debnárová, Simona ; Vojtová, Lucy (oponent) ; Michlovská, Lenka (vedoucí práce)
Biodegradable synthetic polymers bring many advantages over other materials for the utilization in the field of regenerative medicine and tissue engineering. The most important advances involve the capability of optimalizing mechanical or chemical properties and the degradation kinetics. Especially polyesters are interesting because of their simple biodegradation. They undergo the hydrolysis of ester linkage and the degradation products are metabolized without harmful effects. Diploma thesis is focused on synthetic biodegradable PLGA-PEG-PLGA triblock copolymers based on poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and poly(ethylene glycol) (PEG) that belong to the group of biodegradable polyesters. The content of hydrophilic and hydrophobic parts of polymer chain induces the amphiphilic character. Prepared triblock copolymers are capable of forming hydrogel by physical cross-linking in consequence of their amphiphilic character. These materials have noticed significant interest in the field of medical sciences. Theoretical part describes hydrogels, physical cross-linking of amphiphilic block copolymers and the mechanisms of degradation. Description of PLGA-PEG-PLGA triblock copolymers is divided on PLGA copolymers, PEG and their physico-chemical properties. Reliable knowledge of chemical functionalization by succinic anhydride, folic acid and itaconic anhydride is presented. Dopamine is introduces as a linker and the most important bioactive substances are mentioned. Experimental part presents certain methods of synthesis that lead to functionalization and modification of PLGA-PEG-PLGA triblock copolymers. Functionalization by itaconic anhydride was proceeded to obtain functionalized copolymer with both ends capped by reactive double bonds and carboxylic groups. The double bonds enable to form chemical cross-links and the end-capped carboxylic groups offer the opportunity to modify it by biologically active compounds. The modification by bioactive substances L-lysine and butylamine enriches the polymer network and linker dopamine provides the versatility of attached bioactive substances, their stabilization and the maintenance of its biological activity. Final products were characterized by the means of 1H NMR, FTIR and DRA analysis. Functionalization was carried out in a bulk with higher amount of bonded itaconic acid 79.4 mol % and subsequent modifications were proceeded in aqueous solution, organic solution or in a bulk. The most effective method of modification was synthesis in organic solution with solvent N,N-dimethylformamide with activating system dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine. The highest amount of bonded dopamine was 18.6 mol %, the highest amount of attached butylamine was 7.8 mol % and L-lysine was not bonded at all.
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The effect of biologicaly active substances on the structure and properties of collagenous substrates
Muchová, Johana ; Michlovská, Lenka (oponent) ; Vojtová, Lucy (vedoucí práce)
The thesis deals with the preparation of 3D porous collagen scaffolds by freeze-drying and their modification with bioactive compounds. The natural polysaccharides, chitosan, calcium oxidized cellulose and chitin/chitosan-glucan complex for the modification have been used. The mechanical properties of the scaffolds have been enhanced by crosslinking process with carbodiimides. Growth factors have been delivered in the form of platelet lysate. The influence of biologically active additives, crosslinking agents, and enrichment with growth factors on the properties of the prepared scaffold and their bioactivity in tissues of living organisms have been investigated. Specifically, this study includes the morphological properties, structure, porosity, swelling stability, chemical composition, temperature of denaturation and biological properties. Scanning electron microscopy, infrared specktroscopy, differential scanning calorimetry and confocal microscopy have been used to the characterization. Prepared collagen substrates involving bioactive additive and platelet lysate could be used as scaffold for growing cells in systems with low mechanical loading and which has potential application in biomedicine.
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Optimization of antibacterial properties of polymer-phosphate bone fillers
Grézlová, Veronika ; Michlovská, Lenka (oponent) ; Vojtová, Lucy (vedoucí práce)
Proposed diploma thesis is focused on the preparation of polymer-phosphate bone cement. The aim is to optimize antibacterial properties of the cement by adding selenium nanoparticles (SeNPs). The theoretical part describes the characteristics of bone, properties od tricalcium phosphate (TCP) and its polymorphs, the use of bone cement and antibacterial nanoparticles in medicine. The experimental part deals with the preparation of samples, description of the used methods and evaluation of the effect of SeNPs on the bone cement setting reaction, morphology, crystallinity, mechanical, rheological and antibacterial properties. As a result, SeNPs improved bone cement injectability and increased setting reaction with the positive effect on cement mechanical properties. Antibacterial properties of samples were tested using both disk and dilution method resulting in the positive inhibition effect of SeNPs on gram-positive bacteria (G+), especially Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. The quantitative SeNPs release from modified polymer-phosphate bone cement enable its use as antibacterial bone filler (e.g. for osteomyelitis treating).
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Catecholamine modified polymer-composite bone cement: preparation, characterization and in-vitro evaluation
Raszková, Alena ; Vojtová, Lucy (oponent) ; Michlovská, Lenka (vedoucí práce)
This bachelor thesis is focused on preparation, characterization of catecholamine-modified calcium phosphate cement and evaluation of in-vitro tests. The theoretical part deals with the characterization of calcium phosphate cements and their material property. This part also includes overview of additives used to modify cements in order to improve both their chemo-physical and biological properties. The increasing use of catecholamine in tissue engineering is also discussed here. First part of experimental work focuses on optimization of preparation and characterization of catecholamine modified calcium phosphate cement. The phase composition kinetics was evaluated by X-ray diffraction analysis and chemical composition by Fourier-transformed infrared spectroscopy. The second part of the work is then focused on the optimization and evaluation of in-vitro cytotoxicity of modified cements. In conclusion, it was found that the conversion rate during curing was faster when catecholamine was used to prepare phosphate cement. Modified cements exhibit almost 93 % conversion after 3 days with respect to unmodified one where the conversion reach only 79 % at the same time. For in-vitro testing, the new method for preparation of samples was approached. It was found that further optimization of the preparation method and test conditions are necessary for the successful in-vitro tests. Due to attractive properties, catecholamine-modified calcium phosphate cement seems to be an interesting material for bone regeneration.
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