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Preparation of microfiber structures based on PHB copolymer
Kecíková, Alžbeta ; Brtníková, Jana (oponent) ; Přikryl, Radek (vedoucí práce)
Master’s thesis deals with the preparation of microfibrous structures based on poly(3-hydroxybutyrate) (P3HB) and its copolymer with poly(4-hydroxybutyrate) using centrifugal spinning technology. The microfibers were modified by the addition of oligomer P3HB and a plasticizer. The centrifugal spinning process was optimized for each material by solution viscosity, polymer molecular weight, speed of spineret, and presence of solvent. One of the part of optimalization was the addition of formic acid and acetic acid to the dissolution system. Microfibrous structures have been investigated in terms of morphology, mechanical properties, wetting and biocompatibility. P3HB fibers were also surface treated with lipase solutions to reduce their hydrophobicity. The prepared bulky fiber cocoons have a suitable 3D microstructure for monitoring and testing biological properties in vitro. Thus, a potential application of microfiber structures is as 3D cell culture carriers in an scaffolds in vitro system.
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Novel Antibacterial Collagen Scaffolds for Regenerative Medicine
Dorazilová, Jana ; Brtníková, Jana (oponent) ; Vojtová, Lucy (vedoucí práce)
This master’s thesis deals with the characterisation of 3D porous collagenous sponges enriched with selected antibacterial agents. The literature part of the thesis focuses on the overview of biomaterials and biopolymers with the emphasis on collagen and chitosan, outlines the antibacterial properties of nanoparticles and reviews current aspects of using selenium nanoparticles as an antibacterial agent. For the purpose of this work, two types of antibacterial additives were used – biopolymeric chitosan and selenium nanoparticles. Preparation of 3D porous structure was achieved using the freeze drying method. Mechanical properties of prepared biopolymeric matrices were improved by chemical crosslinking in the presence of carbodiimide. Predominantly physiochemical methods were used for characterization of prepared collagenous sponges. For microstructure analysis, pore size determination, visualisation of nanoparticles and their distribution inside the porous structure, scanning electron microscopy (SEM) with energy dispersive x ray optical analysis (EDX) was used. Parameters such as total porosity, swelling ratio, weight loss during degradation in water and enzymatic environment were evaluated by suitable gravimetric methods. Fourier transformed infrared spectroscopy with attenuated total reflectance (ATR-FTIR) was used to determine changes in the chemical structure of collagenous matrices before and after addition of the antibacterial agents. Percentage release of nanoparticles was evaluated using optical emission spectroscopy with inductively coupled plasma (ICP OES). Evaluation of antibacterial properties of tested samples was carried out mainly by the agar diffusion disk method and the macrodilution broth method. In the conducted research we were able to determine the influence of selected antibacterial additives on the physiochemical properties of 3D collagenous matrices. Their antibacterial activities showed a positive effect on bacterial inhibition of both chitosan and selenium nanoparticles with respect to their concentrations. The designed materials could be further utilized for bio medicinal applications, especially in the field of soft tissue regeneration.
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Optimization of methods for protein analysis released from thermosensitive hydrogel
Lysáková, Klára ; Brtníková, Jana (oponent) ; Vojtová, Lucy (vedoucí práce)
Proposed diploma thesis is focused on the release evaluation methods of both model proteins (albumin and lysozyme) and tissue healing protein (stable fibroblast growth factor 2; FGF2-stab) from the “smart” hydrogel carrier based on the biodegradable thermosensitive PLGA-PEG-PLGA triblock copolymer. In the theoretical part, a brief overview of thermosensitive polymers, their properties, structure and utilization of aforementioned proteins is described. Moreover, types of interactions that may occur between proteins and the polymeric carrier including drug release models are mentioned. In the experimental part, the structure of PLGA-PEG-PLGA triblock copolymer was characterized by the gel permeation chromatography and 1H NMR while its visco-elastic properties including sol-gel transition were evaluated by the rheological analysis. Thesis goal was targeted to better understand the release of newly-patented FGF2-stab protein from injectable PLGA-PEG-PLGA hydrogel formed at physiological temperature. The amount of released FGF2-stab was measured by the UV-VIS spectrophotometer in the presence of a Bradford reagent that binds proteins resulting in shifting their absorption maxima from 280 nm to 595 nm. For the comparison, SDS-page electrophoresis, dividing protein by molecular weight, has been used. It has been find out, that model proteins, which were in different size but predominantly non-polar on their surface exhibited two-stage release dependent on both the diffusion and polymer degradation, whereas, FGF2-stab (25 kg.mol-1) showed controlled one-stage first-order release from the PLGA-PEG-PLGA hydrogel forced only by diffusion, since it is predominantly a polar molecule located probably at the hydrogel micelle surface and not in their core. These results are very important to tune the protein release from hydrogel carriers to meet their certain application, in this case specifically in controlled tissue regeneration.
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Effect of bioceramic additives on morphology, physical and biological properties of collagen scaffolds for bone tissue engineering
Klieštiková, Nikola ; Poláček, Petr (oponent) ; Brtníková, Jana (vedoucí práce)
The diploma thesis deals with preparation of three-dimensional porous collagen composite scaffolds for bone tissue engineering and study of the effect of addition of bioceramic particles on morphological, biomechanical and biological properties. Theoretical part describes biomaterials and bioceramic particles used for scaffolds in bone tissue engineering and their fabrications method. As for experimental part, samples were prepared by the freeze-drying method. As tested material, type I collagen from porcine and bovine sources was combined with hydroxyapatite and mixture of -tricalcium phosphate and -tricalcium phosphate in ratios 1 : 1, 1 : 2 and 2 : 1. The effect of bioceramics solubility and particle sizes on scaffolds morphology, biomechanics and biocompatibility was evaluated. Addition of bioceramic particles changed the morphology of the samples. The pore size decreased, whereas the porosity was nearly the same in all tested samples. Bioceramic particles also made the collagen matrix of the scaffolds less hydrophilic, moreover they stabilized the scaffolds against the effect of enzymatic degradation. The biomechanical properties of the samples were tested in both dry and hydrated state. In dry state, the pure bovine collagen scaffolds reached the highest compressive strength, contrary in hydrated state, the samples containing bioceramic particles reached the highest value. None of the samples was cytotoxic and the most preferable environment for cell adhesion and proliferation was in the pure bovine collagen scaffolds and also in the composite scaffolds with ratio HAp : -TCP : 1 : 1.
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The effect of salts on the hydrolytic stability of biopolymer carriers with antibacterial substances
Tertinská, Martina ; Brtníková, Jana (oponent) ; Vojtová, Lucy (vedoucí práce)
This diploma thesis deals with observing the effects of salt on the stability of biopolymer carriers made of protein and polysaccharide. The theoretical part is concerned with biomaterials and materials for sample preparation and also deals with the current situation of treatment of infected wounds with various materials. Within the experimental part, the carriers were prepared in two different buffers with different concentrations of sodium chloride in order to stabilize bioactive proteins, peptides or enzymes generally used for carrier entrapment. In every series of samples there were prepared both crosslinked and non-crosslinked samples. Properties like hydrolytic stability and swelling ratio were tested and the effect of salt concentration on sample stability was observed. Since the optimal salt concentration has been established, antibacterial enzyme was entrapped into all carriers and its release was monitored. Whereas at non-crosslinked samples significant differences between salt and salt-free samples both stability and enzyme release have been monitored, at crosslinked samples the differences were neglectable. However, in all cases the enzyme released from foamed samples according to first order kinetics, which is concentration dependent. Based on the results from disk diffusion test provided on different strains of S. aureus, the concentration of 325 mg/ml, have been chosen for my further work. Prepared carriers with antibacterial enzyme could be used as both- fast or prolonged releasing wound dressings for Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA) infected tissue.
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Porous hydrogels from natural gum modified with bio-active substances for accelerated wound healing
Černá, Eva ; Brtníková, Jana (oponent) ; Vojtová, Lucy (vedoucí práce)
The main aim of this thesis is to design, prepare and optimize composition of hydrogel dressing for wound healing with skin-like properties. An inexpensive hydrogel dressing with properties that accelerate healing process, supress bacterial infection and support easy handling is supposed to be a result of this work.. The theoretical part is focused on properties of each layer of the skin, polymer materials suitable for wound healing, pro-healing and antimicrobial agents suitable for antimicrobial environment in the wound. The experimental part includes several steps. The first one is optimization of the composition of the porous hydrogel dressing composed of natural polymer, resin Gum Karaya (GK) modified with synthetic hydrophilic gelling polymers and important emollient componentto obtain soft, elastic and transparent films via freeze-drying technique. Films were tested in terms of swelling ratio, hydrolytic stability, transparency, strength in tension in both dry and wet conditions, chemical composition and morphology. The second step includes integration of bioactive antiseptic compound into the structure of the hydrogel, which provides suitable antimicrobial properties for wound healing. Prepared transparent, hydrolytically stable and very tough hydrogel films were found to be effective against both gram-positive and gram negative bacterial strains tested within these work, specifically Staphylococcus aureus methicillin-sensitive, Staphylococcus aureus methicillin-resistant, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae, Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans and Escherichia coli. Followed work will be focused on both in vitro and in vivo biocompatibility evaluations.
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The effect of organic and inorganic additives on the chemico-physical properties of biopolymer substrates for tissue engineering
Kohoutek, Martin ; Muchová, Johana (oponent) ; Brtníková, Jana (vedoucí práce)
This bachelor’s thesis deals with the preparation and characterization of composite collagenous scaffolds for possible applications in both bone and skin tissue engineering with an emphasis on their chemico-physical properties. In the theoretical part, the selected components for fabrication of the scaffolds are described and later were used to fabricate 3D porous composite scaffolds in the experimental part. Altogether, four different composite collagenous scaffold types and a reference pure collagen scaffold type were prepared using the freeze-drying fabrication technique. Two scaffold types were made by combining collagen with either oxidised cellulose (OC) or carboxymethyl cellulose (CMC). The other two types of scaffolds had the same biopolymeric origin enhanced with the addition of bioceramics based on the hydroxyapatite and tricalcium phosphates. The microstructure, porosity and pore size were assessed by the scanning electron microscopy. The highest porosity and pore size were achieved by the reference purely collagenous scaffolds, followed by the collagen composites with OC and CMC. Scaffolds with the content of bioceramics had the lowest porosity and pore size, especially those containing CMC. Swelling behaviour analysis and enzymatic degradation in vitro showed, that the hydrophilicity and mass loss in the degradation process correlate with each other. The scaffolds without bioceramics were more hydrophilic and achieved greater mass loss than the scaffolds containing bioceramics. The pure collagen was in the between the two groups. Scaffolds containing CMC achieved greater mass loss and hydrophilicity than their OC counterparts. In terms of mechanical properties, scaffolds with bioceramics achieved higher compressive strength in the wet state than the other three scaffold types. The mechanical properties were generally better for scaffolds with lower porosity and lower hydrophilicity.
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Preparation and characterisation of encapsulated biogenic nanoparticles for medical application
Poláková, Veronika ; Vojtová, Lucy (oponent) ; Brtníková, Jana (vedoucí práce)
The aim of this bachelor thesis is a preparation and polymeric encapsulation of antibacterial biogenic nanoparticles in order to enhance their stability, reduce possible cytotoxicity while maintaining antibacterial activity. The theoretical part contains an overview of regenerative medicine, commonly used nanostructures in regenerative medicine, their properties, and methods of encapsulation. The experimental work especially focuses on selenium nanoparticles synthesis using different methods with specific protecting agents followed by encapsulation via nature-inspired polymer. The used encapsulation methods are based on self-assembly polymerization and coating of selected natural polymeric adhesive. The chemical and physical properties of pure and encapsulated selenium nanoparticles, such as their concentration and morphology (size and shape) were studied using Fourier transformed infrared spectrophotometry and scanning transmission electron microscopy. It was found that different used method provides nanoparticles with different size, shape and stability. As a main result, an optimized method of selenium particles synthesis, stabilization and encapsulation was developed and described. Nanoparticles, synthesized using this method, are spherical with size ranging from 10.5 to 101 nm. The sizes of most of the synthesized nanoparticles lay within 10.5 to 40 nm interval. When encapsulated, their sizes increase and are ranging from 74.5 to 571.5 nm.
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Fotostabilization of biopolymeric materials for regenerative medicine
Izsák, Dávid ; Muchová, Johana (oponent) ; Brtníková, Jana (vedoucí práce)
Non-stabilized biopolymers often show poor mechanical properties. The stabilization and improvement of these properties could be accomplished by crosslinking, but the traditionally used crosslinking agents are rather cytotoxic and harmful for the human body. The aim of this work is to find a suitable non-toxic crosslinking method that would be efficient enough, but at the same time would not represent danger to any organism. The crosslinking method developed in the presented bachelor thesis should be comparatively efficient as traditionally used methods (e.g. chemical carbodiimide-based crosslinking) as well as potentially less cytotoxic by use of naturally derived substances. The proposed alternative crosslinking method should improve the strength and stability of the crosslinked biopolymers and enhance their biological functionality. Specifically, the presented work deals with the stabilization of collagen scaffolds using vitamin important for human metabolism followed by short- and long-wavelength irradiation. The theoretical part contains a general overview of possible non-toxic crosslinking agents, describes the structure of collagen and characterizes its properties and with the properties of used vitamin as a crosslinking agent. The experimental part deals with the preparation and crosslinking optimization of collagen scaffolds. Swelling, degradation and morphology of scaffolds as well as collagen secondary structure were evaluated. As a result, sufficiently strong crosslinked scaffolds were obtained.
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