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Development of antimicrobial wound healing nanostructure biomaterials
Hanák, Jan ; Wikarská, Monika (referee) ; Márová, Ivana (advisor)
The bachelor thesis is focused on optimizing the production of nanoparticles with a bioactive component - especially on enzymes and antibiotics encapsulated into lipozomes. Nanoparticles with active substances could be used in the field of pharmacy or cosmetology. In the theoretical part the composition of the skin, skin injuries, and wound covers are described. The materials used for the preparation of nanoparticles and nanofibers are characterized as well as processes to produce such nanomaterials. Enzymes, antimicrobial drugs, and methods for determining antimicrobial activity are mentioned in this section, too. The practical part deals with the characterization and optimization of the formation of lipozome particles with encapsulated enzymes. The formed particles were divided into two groups. The first type of particles were pure lipozomes, while the second ones enriched by polyhydroxybutyrate to increase the encapsulation efficiency and improve the stability of the particles. The enzymes bromelain and lysozyme were selected for the highest encapsulation efficiency. The encapsulation of these two enzymes took place in two ways - by thin film hydration and by sonication method. Both methods showed high encapsulation efficiency, but the sonication method proved to be unsuitable due to enzyme denaturation. The proteolytic activity of the released enzymes from the particles was measured. The stability of the particles was monitored in a physiological and aquatic environment by measuring the zeta potential for up to 28 days. Moreover, the enzymes, ampicillin, and particles created by sonication were subsequently subjected to antimicrobial tests by using two test bacterial strains. Finally, alginate particles with encapsulated enzymes were also formed, then the encapsulation efficiency and proteolytic activity of the enzyme were measured. In this study also other methods of encapsulation of active substances are outlined, which could be possibly used to produce nanomaterials suitable for wound healing.
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Modification of hydrogel wound dressings by biological active agents
Štrublová, Veronika ; Říhová, Martina (referee) ; Vojtová, Lucy (advisor)
Cílem práce je navrhnout a připravit cenově výhodný hydrogelový kryt ran, který bude chránit ránu před mikrobiální infekcí a urychlovat proces hojení. Teoretická část shrnuje jak přírodní, tak syntetické polymery, antibakteriální a pro-hojivá aditiva, která jsou široce používána v biomedicínských aplikacích. Obsahuje také krátký přehled jednotlivých fází hojení ran. Experimentální část předložené práce se zabývá přípravou a charakterizací hydrogelových krytů ran s porézní strukturou složených jak ze syntetických, tak přírodních polymerů zahrnujících přírodní polysacharid Gum Karaya (GK), polyvinylalkohol a polyethylenglykol. Připravené lyofilizované hydrogelové kryty ran byly obohaceny biologicky aktivními látkami – antibakteriálními nanočásticemi selenu a fibroblastovým růstovým faktorem FGF2, který představuje protein pro hojení ran. Pro charakterizaci lyofilizovaných hydrogelových krytů ran byla použita řada fyzikálně-chemických technik. Metoda zeslabeného úplného odrazu infračervené spektroskopie s Fourierovou transformací (ATR-FTIR) byla použita ke stanovení změn v chemické struktuře, biomechanické vlastnosti za fyziologických podmínek byly vyhodnoceny pomocí tahové zkoušky za použití dynamicko-mechanické analýzy, morfologie byla charakterizována skenovací elektronovou mikroskopií (SEM) a botnací vlastnosti připravených vzorků byly zjištěny vhodnou gravimetrickou metodou. K vyhodnocení antimikrobiálních vlastností GK byla použita jak mikrodiluční bujónová metoda, tak difúzní agarová disková metoda. Cytotoxicita bylo kvantifikována na základě A) počítání počtu živých a mrtvých buněk v extraktech z GK a B) měřením buněčné proliferace pomocí resazurinové eseje. In vivo testování proběhlo ve spolupráci s Výzkumným ústavem veterinárního lékařství na praseti bílém jako animálním modelu. Z výsledků vyplynulo, že vzorky obsahující glycerol vykazují vynikající mechanické vlastnosti jak za suchého, tak za mokrého stavu. Výsledky antibakteriálních testů ukázaly pozitivní synergický efekt GK a selenových nanočástic na inhibici růstu všech testovaných G+ kmenů. In vivo a in vitro testy prokázaly zvýšenou hojivou schopnost připravených materiálů bez způsobení cytotoxických reakcí. Nové porézní a velmi stabilní hydrogelové kryty ran jsou nadějnými materiály, které mohou být využity v biomedicínských aplikacích, především v oblastech vlhkého hojení ran.
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Encapsulation of active substances into nanofibers and possibilities of their application
Procházková, Lucie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
The master thesis was based on the optimization of the production of nanofiber covers and to gaine the product for subsequent functional use. The production of nanofiber covers was made by electrospinning and forcespinning from selected materials. Polyhydroxybutyrate, gelatin, chitosan and alginate were used as starting materials. After successful optimization, these materials were enriched with active ingredients ampicillin and ibuprofen for the functionalized use of covers for more effective wound healing. The theoretical part was focused on the issue of skin, healing processes, types of wounds and nanofibers, the characterization of selected starting materials for the formation of nanofibers was also mentioned. The practical part was based on the lengthy optimization of the preparation of fiber covers and later enriched with active ingredients. Furthermore, combined covers made of different materials with contents of both active ingredients were designed. This was followed by the characterization of all prepared covers from the point of view of stability in the short and long term. The gradual release of active ingredients was determined spectrophotometrically and by hifh performance liquid chromatography. It was also important to determine the antimicrobial activity of selected active substances. At the end of all testing, combined coatings containing both active ingredients were used for safety testing with human keratinocyte cells (HaCaT). Safety testing was based on determining the viability of human cells using the MTT test, to verify the LDH test. A scratch test was also performed, a wound healing test after the application of devised combined covers.
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Role of fibroblasts in wound healing and cancer
Mateu Sanz, Rosana ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Mokrý, Jaroslav (referee)
Fibroblasts are stromal cells ubiquitously present in the human body. They often appear in a quiescent state and can become activated in response to tissue remodeling signals. Activated fibroblasts acquire biosynthetic, pro-inflammatory and contractile properties, key functions for wound healing. In addition, the presence of permanently activated fibroblasts is one of the hallmarks of cancer. The purpose of this work is to investigate the differences between newborn and adult fibroblasts and keratinocytes in their implication in scarless wound healing, the origin of cancer associated fibroblasts (CAF)s and the influence of fibroblasts in melanoma invasion. Evidence suggests that wounds heal almost without scar in newborns. To understand the mechanisms that contribute to scarless wound healing we focused on the differences between newborn and adult fibroblasts and keratinocytes, which are cells present in human skin and participating in wound healing process. A comparison of the expression profile between newborn and adult fibroblasts showed differentially regulated genes related to the acute phase of the inflammatory response and ECM organization, traits involved in wound healing. We also found that newborn fibroblast showed higher differentiation potential, exhibited markers of pluripotency and...
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Study of plasma interaction with bacteria for wound healing
Šrámková, Sarah ; Krčma, František (referee) ; Kozáková, Zdenka (advisor)
This thesis focuses on the study of the interaction of plasma with bacteria to enhance the promotion of wound healing. Firstly, the wound healing process is described, followed by low temperature plasma, its effect on bacterial cells and its use in biomedicine. The experimental part deals with the application of low-temperature plasma generated by microwave jet on selected microorganisms and the influence of experimental conditions on the antimicrobial effect of the plasma. One representative of the microorganisms occurring in the wounds was selected as representatives of Gram-positive bacteria and Gram-negative bacteria, namely Staphylococcus epidermidis and Escherichia coli. Using the results obtained, the antimicrobial effect of plasma generated by microwave jet in argon was confirmed. The degree of antimicrobial effect is related to the concentration of the microorganisms, the time of treatment and whether the bacteria are Gram-positive or Gram-negative.
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Role of matrix metalloproteinases in regulation of late embryonic healing process
Kikinderová, Paulína ; Šindelka, Radek (advisor) ; Procházka, Jan (referee)
Cutaneous wound healing could be distinguished into two main types: embryonic and adult. Embryonic healing in contrast to adult is faster, scar-less and consists of early, middle, and late phases. Actin ring is formed during the early phase and its cables pull the edges of the wound towards apposite sites during the following middle phase. De novo expression of healing specific genes is initiated also during middle phase. However, process of the wound healing continues under the closed wound in the late phase which has been poorly described. Adult wound healing is more complex, longer, and is divided into 4 phases: haemostasis, inflammation, proliferation, and remodelling phase. Adult wound healing might end with the scar. Pivotal role in the wound healing is given to matrix metalloproteinases (MMPs). These remodelling enzymes are important for releasing cytokines, inducing apoptosis, and degradation of extracellular matrix. Our laboratory performed temporal RNA-sequencing of the healing tissue using tailbud stage and swimming tadpole embryos. Results showed predominant expression of four mmps: mmp1.L, mmp7.S, mmp8.S, and mmp9.L. Injury or amputation caused the upregulation and their expression level peaked at 3-6 hours post injury which corresponds with late phase of healing. Thus, I focused on...
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Use of plasma jet for wound healing
Dvořáková, Eva ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
This diploma thesis was focused on the possibility of using a plasma nozzle to accelerate the wound healing process. The benefits of using low-temperature plasma in medicine or biomedical applications are known from many studies, and low-temperature plasma is already used to sterilize medical devices, materials or surgical instruments. Some studies also report a high potential of usinh plasma nozzle in the treatment of skin wounds. In the experimental part of this work, an in vitro wound healing test was performed using two different low-temperature plasma sources. Source No. 1 was a surface wave microwave discharge and source No. 2 was a torch microwave discharge. An in vitro scratch healing test was performed on a monolayer of HaCaT keratinocytes and testing was performed using various parameters. The influence of the plasma treatment time was monitored, as well as the influence of the plasma discharge power and also the influence of the argon working gas flow. Especially when using a torch microwave discharge, faster wound healing was recorded at most of the parameters used compared to the control. Thus, it can be said that this source appears to be potentially suitable for faster wound healing. Furthermore, in the work using the MTT cytotoxicity test, the viability of skin cells after their plasmination was also monitored using the same conditions as in the in vitro wound healing test. When performed in the standard MTT assay, none of the settings or sources used showed any cytotoxic effects on keratinocytes. LDH cytotoxicity tests were also performed concurrently to verify the accuracy of the MTT assays. The results of both tests agreed and the use of low-temperature plasma in skin treatment can be considered as safe. Overall, the results show that the plasma nozzle can find use in medicine in the healing of skin wounds and chronic defects as a potentially fast, inexpensive and effective method.
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