Syntéza a funkcionalizace uhlíkatých nanomateriálů pro tkáňové inženýrství
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Technická Univerzita v Liberci
Abstract
Výzkum materiálů vhodných pro tkáňové inženýrství patří mezi rychle se rozvíjející oblasti; pro kultivaci buněk jsou, mimo jiné, úspěšně požívány různé uhlíkaté materiály. Bylo prokázáno, že adheze a proliferace buněk závisí na mnoha faktorech, jako například přítomnost určitých funkčních chemických skupin, smáčivost povrchu, jeho povrchová energie, morfologie, mechanické vlastnosti apod.Tato práce představuje nový typ funkcionalizace, při němž byl povrch uhlíkatých nanomateriálů modifikován amid-aminovými skupinami. Velikost a struktura částic byly zkoumány transmisní elektronovou mikroskopií. Specifický měrný povrch a objem pórů byl stanoven pomocí dusíkové adsorpce a vypočten z Brunauer-Emmet-Tellerovy rovnice. Chemické složení připravených materiálů bylo zkoumáno prostřednictvím rentgenové fotoelektronové spektroskopie a organické elementární analýzy, teplotní stabilita pomocí diferenční snímací kalorimetrie.Funkcionalizované částice byly navázány na povrch polymerních povrchů z polyethylentereftalátu a vysokohustotního polyethylenu, aktivovaných argonovým plasmováním. Chemické složení modifikovaných povrchů bylo analyzováno pomocí Ramanovy a rentgenové fotoelektronové spektroskopie a měřením elektrokinetického potenciálu. Drsnost povrchu byla měřena pomocí mikroskopu atomárních sil, jeho smáčivost pak měřením kontaktního úhlu. Adheze a proliferace potkaních hladkosvalových buněk na modifikovaných površích byla zkoumána in vitro.Výše zmíněné funkcionalizované uhlíkaté nanočástice byly dále využity k přípravě kompozitních nanovlákenných nosičů z polykaprolaktonu. Tyto materiály pak byly porovnávány s čistými polykaprolaktonovými nanovlákny a nanovlákenným kompozitem, obsahujícím pouze neupravené uhlíkaté částice. Struktura připravených nosišů byla zkoumána skenovacím elektronovým mikroskopem, specifický měrný povrch stanoven pomocí dusíkové a kryptonové adsorpce. Cytokompatibilita byla studována v rámci MTT testování na linii 3T3 myších fibroblastů. Vzorky byly dále obarveny pro fluorescenční mikroskopii, během níž při ozařování modrým a zeleným světlem došlo u vzorků obsahujících uhlíkaté částice k tepelnému natavení nanovlákenné polymerní matrice. Nosiče s funkcionalizovanými uhlíkatými částicemi vykazovaly mnohem vyšší cytokompatibilitu než vzorek s neupravenými částicemi. Dále byly testovány antibiotické vlastnosti nosiče obsahujícího neupravené částice (v porovnání s čistými polykaprolaktonovými nanovlákny) za použití bakteriálních kmenů Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis. Žádná antibakteriální aktivita nebyla prokázána.Podstatná část práce je též zaměřena na syntézu mezoporézních uhlíkatých materiálů pomocí soft a hard templátování. Připravené materiály byly analyzovány pomocí skenovací elektronové mikroskopie a dusíkové adsorpce. Vybrané materiály byly dále funkcionalizovány výše zmíněným způsobem a charakterizovány za použití organické elementární analýzy a dusíkové adsorpce. Tyto materiály jsou určeny pro budoucí experimenty s navazováním částic na polymerní povrchy pro tkáňové inženýrství.Vzhledem k interdisciplinární povaze této studie se její autorka snažila osvojit si co nejvíce užitých metod, aby byla schopna vytvořit komplexní práci. Zaměřila se tedy nejen na syntézu a funkcionalizaci uhlíkatých částic, ale také na přípravu nanovlákenných materiálů, plasmování, analýzu specifického měrného povrchu a porosity plynovou adsorpcí, základy tkáňového inženýrství a fluorescenční mikroskopie. Aktivně se podílela na převážné většině provedených experimentů a analýz.
Research of materials suitable for tissue engineering is a rapidly developing area. Among many other, various carbon materials have been successfully used for cell cultivation. There are a lot of factors that have been proved to affect cell adhesion and proliferation, such as the presence of various chemical groups, wettability of the surface, surface energy, morphology, mechanical properties etc.In this work, a novel type of functionalisation was performed, in which carbon nanoparticles (CNPs) were successfully functionalised with amide-amine groups. The size and form of the CNPs was investigated by transmission electron microscopy (TEM). The specific surface areas and pore volumes of the particles were measured via nitrogen adsorption and calculated from Brunauer-Emmet-Teller (BET) equation. The chemical composition was studied via X-ray photoelectron spectroscopy (XPS) and organic elemental analysis (OEA). Differential scanning calorimetry (DSC) was used to investigate thermal stability of CNPs.The functionalised CNPs were grafted onto the surface of polyethylene terephthalate (PET) and high density polyethylene (HDPE) pre-activated in argon plasma. The chemical composition of the modified polymer surfaces was determined by Raman and X-ray photoelectron spectroscopies and by electrokinetic analysis (zeta potential). Surface roughness and morphology of polymers grafted with CNPs was studied by atomic force microscopy (AFM), surface contact angle was measured by goniometry. Adhesion and proliferation of rat vascular smooth muscle cells (VSMC) on HDPE and PET surfaces grafted with functionalised CNPs were studied in vitro.Using the aforementioned CNPs, four types of composite scaffolds were prepared via sputtering the CNPs into electrospun polycaprolactone (PCL) nanofibers: three of them with three types of functionalised CNPs and one with plain activated CNPs. Plain PCL nanofibers and the composite nanofibrous scaffold with plain activated CNPs were used as comparative samples. The structure of the materials was studied using scanning electron microscopy (SEM). The specific surface area of the scaffolds was measured via nitrogen and krypton adsorption and calculated from BET equation. Cytocompatibility of the materials was tested using 3T3 mouse fibroblasts. Cell viability and proliferation was measured by MTT assay on days 1, 3, 8 and 14 after cell seeding. The samples were then stained using fluorescent dyes and examined via fluorescence microscopy (FM). During the FM analyses, all scaffolds containing CNPs underwent structural degradation when irradiated with either green or blue light. The scaffolds with functionalised CNPs showed better cytocompatibility than the scaffold with plain CNPs. The PCL scaffold with plain CNPs was also tested for antibacterial activity (in comparison with plain PCL nanofibers) using the following bacterial strains: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis. However, no antibacterial effect was found.A substantial part of the thesis is also focused on the synthesis of mesoporous carbon particles via soft- and hard-templating. The materials were analysed using SEM and gas adsorption analyses. Selected synthesised materials were functionalised using the above mentioned amide-amine functionalisation and characterised using OEA and gas adsorption. These materials are to be used for grafting on plasma treated polymer surfaces.As this study is highly interdisciplinary, the author wanted to manage as many procedures as possible to make a complex piece of work. She therefore focused on being trained not only in synthesis, but also production of fibrous materials, plasma treatment, gas physisorption, basics of tissue engineering and fluorescence microscopy. Not only did she use the skills gained to prepare the materials, but also actively participated in the majority of the procedures and analyses performed throughout the study.
Research of materials suitable for tissue engineering is a rapidly developing area. Among many other, various carbon materials have been successfully used for cell cultivation. There are a lot of factors that have been proved to affect cell adhesion and proliferation, such as the presence of various chemical groups, wettability of the surface, surface energy, morphology, mechanical properties etc.In this work, a novel type of functionalisation was performed, in which carbon nanoparticles (CNPs) were successfully functionalised with amide-amine groups. The size and form of the CNPs was investigated by transmission electron microscopy (TEM). The specific surface areas and pore volumes of the particles were measured via nitrogen adsorption and calculated from Brunauer-Emmet-Teller (BET) equation. The chemical composition was studied via X-ray photoelectron spectroscopy (XPS) and organic elemental analysis (OEA). Differential scanning calorimetry (DSC) was used to investigate thermal stability of CNPs.The functionalised CNPs were grafted onto the surface of polyethylene terephthalate (PET) and high density polyethylene (HDPE) pre-activated in argon plasma. The chemical composition of the modified polymer surfaces was determined by Raman and X-ray photoelectron spectroscopies and by electrokinetic analysis (zeta potential). Surface roughness and morphology of polymers grafted with CNPs was studied by atomic force microscopy (AFM), surface contact angle was measured by goniometry. Adhesion and proliferation of rat vascular smooth muscle cells (VSMC) on HDPE and PET surfaces grafted with functionalised CNPs were studied in vitro.Using the aforementioned CNPs, four types of composite scaffolds were prepared via sputtering the CNPs into electrospun polycaprolactone (PCL) nanofibers: three of them with three types of functionalised CNPs and one with plain activated CNPs. Plain PCL nanofibers and the composite nanofibrous scaffold with plain activated CNPs were used as comparative samples. The structure of the materials was studied using scanning electron microscopy (SEM). The specific surface area of the scaffolds was measured via nitrogen and krypton adsorption and calculated from BET equation. Cytocompatibility of the materials was tested using 3T3 mouse fibroblasts. Cell viability and proliferation was measured by MTT assay on days 1, 3, 8 and 14 after cell seeding. The samples were then stained using fluorescent dyes and examined via fluorescence microscopy (FM). During the FM analyses, all scaffolds containing CNPs underwent structural degradation when irradiated with either green or blue light. The scaffolds with functionalised CNPs showed better cytocompatibility than the scaffold with plain CNPs. The PCL scaffold with plain CNPs was also tested for antibacterial activity (in comparison with plain PCL nanofibers) using the following bacterial strains: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis. However, no antibacterial effect was found.A substantial part of the thesis is also focused on the synthesis of mesoporous carbon particles via soft- and hard-templating. The materials were analysed using SEM and gas adsorption analyses. Selected synthesised materials were functionalised using the above mentioned amide-amine functionalisation and characterised using OEA and gas adsorption. These materials are to be used for grafting on plasma treated polymer surfaces.As this study is highly interdisciplinary, the author wanted to manage as many procedures as possible to make a complex piece of work. She therefore focused on being trained not only in synthesis, but also production of fibrous materials, plasma treatment, gas physisorption, basics of tissue engineering and fluorescence microscopy. Not only did she use the skills gained to prepare the materials, but also actively participated in the majority of the procedures and analyses performed throughout the study.
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Subject(s)
uhlíkaté nanomateriály, funkcionalizace, tkáňové inženýrství, cytokompatibilita, amin, carbon nanomaterials, functionalisation, tissue engineering, cytocompatibility, amine