Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs. Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns. While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.

This diploma thesis was focused on basic physicochemical properties of caffeine and investigation of the interaction between the polysaccharide sodium hyaluronan (HA), pectin (PEC) or lignohumate (HUM) and the alkaloid caffeine (CAF). The reason why this thesis was focused on study of aqueous solutions of hyaluronan or pectin with caffeine is that the presence of polysaccharide- caffeine complex could be beneficial for slowing the absorption of caffeine. In the theoretical part of the diploma thesis there is described a group of alkaloids, which caffeine belongs to and also polysaccharides hyaluronic acid and pectin. The most attention has been paid to caffeine, its properties and effects on the human organism. This diploma thesis also deals with non-alcoholic beverages with caffeine, 4-methylimidazole, and phosphoric acid. The last chapter of the theoretical part is dedicated to fluorescence spectroscopy. In the experimental part of this work I deal with the basic properties of caffeine and interaction between the caffeine and selected polysaccharides or lignohumate. At first, the chemical and physical properties of caffeine were determined. Based on these results the interaction of caffeine and hyaluronan or pectin was studied by using the fluorescence and absorbance. The HA of molecular weight of 1,7 MDa and pectin from citrus fruits were chosen for the study of the interaction with the caffeine. These two substances were mixed with the caffeine of concentration contained in Coca-Cola. The results of interaction that would lead to the influence of the emission or absorption properties of caffeine, were not proved. As a part of the work there was determined how pectin behaves in strongly acidic solution and then there was monitored the pH, conductivity and solubility of caffeine in water by using thermogravimetry and visual experiments. The obtained results of the interaction of hyaluronan-caffeine, pectin caffeine or caffeine-lignohumate can be used for description of the behavior of caffeine in the presence of selected polysaccharides and natural organic substance.

Všeobecne, cieľom tejto diplomovej práce bola príprava a charakterizácia sieťovaných hyaluronových mikrovláken, ktoré by boli stabilné vo vodnom prostredí s vhodnými mechanickými vlastnosťami. Vlákna pripravené technikou zvlákňovania z roztoku boli sieťované pomocou roztoku so sieťovacím činidlom 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimidom alebo roztoku s dvoma sieťovacími činidlami 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimidom a N-hydroxysuccinimidom, vedúcim k amidácii a tvorbe esterových väzieb. Charakterizácia pripravených vzoriek je hlavným cieľom experimentálnej časti práce. Charakteristiky botnání boli vykonané na určenie stability vláken v troch rôznych roztokoch (s pH 7.4, 3 a 11). Na určenie termickej stability bola využitá termogravimetrická analýza a na stanovenie esterifikácie a amidácie bola použitá infračervená spektroskopia s Fourierovou transformáciou. Mechanické vlastnosti vláken boli študované pomocou testovania závislosti stress-strain. Ďalej boli testované reologické vlastnosti ako aj mikroštruktúra a povrch vláken pomocou skenovacieho elektrónového mikroskopu. Vlákna pred chemickým sieťovaním vykazovali nižšiu stabilitu vo všetkých troch roztokoch, termálna stabilita bola taktiež nižšia ako stabilita zosieťovaných vláken. Pre vlákna chemicky nemodifikované so sieťujúcim činidlom, sa objavil iba jeden typ píku pre esterifikáciu. Infračervené spektrum chemicky zosieťovaných vláken ukázalo prítomnosť dvoch píkov pre esterifikáciu, čo je prejavom efektivity sieťovacieho činidla. Amidácia bola tiež výraznejšia pri zosieťovaných vláknach, špeciálne pre vlákna sieťované dlhú dobu a v roztoku s vyššou koncentráciou sieťujúceho činidla. Kvôli nerovnomerným vláknam, mechanické vlastnosti nevykazovali žiadnu závislosť na sieťovaní. Štúdium reológie ukázalo, že viskozita vlákna rozpusteného vo vode je menej závislá na šmykovej rýchlosti ako prášok hyaluronanu sodného rozpusteného vo vode. Napriek mnohým rôznym metódam charakterizácie vláken, ktoré boli použité v tejto práci, je stále veľa možností pre lepšiu charakterizáciu a bližšie pochopenie tohto biopolymérneho materiálu.

This master thesis focuses on the effect of cultivation parameters on hyaluronic acid synthesis during its biotechnological production. The cultivation parameters were temperature of the cultivation, aeration, agitation, carbon source and addition of phosphatidylcholine. Changes in molecular weight and yield of hyaluronic acid, growth of biomass and medium viscosity were observed. From the obtained data we learned, that the specific growth rate greatly impacts final characteristics of hyaluronic acid. Under suboptimal temperatures a high molecular weight polymer was synthesised along with comparable yields from the control cultivation. High temperatures (40 °C) caused a dip in molecular weight. The next cultivation parameters were aeration and agitation. The highest molecular weights were obtained in cultivations with high agitation rates and intensive aeration, specifically 1 vvm/800 rpm and 2 vvm/800 rpm. Agitation had a bigger influence on molecular weight than aeration. When the carbon source was changed from sucrose to glucose, the lowest molecular weight and yield were obtained. Addition of phosphatidylcholine with concentration 160 mg/l had a positive effect on the cultivation, where the molecular weight grew and biomass growth was higher.

A new method for determining the viscoelastic properties of materials was introduced and investigated. Results of three groups of samples obtained using one particle microrheology method, DLS microrheology method and classical rheology method were compared to be sure of correctness of measurements. As a model system were chosen mixtures of glycerol of different viscosities. In case of samples containing glycerol, results were also compared with tabulated values. Hyaluronan of various molecular weights was used as a biopolymer and polystyrene particles were used for microrheology. It was confirmed, that viscosity values of biopolymer samples obtaining by each method are comparable.

Hydrogels are high water-content polymers that mimic the extracellular matrix of cells. The polymers can have many sources and be of natural origin from the extracellular matrix (ECM) of cells or be synthetically derived. Two such polymers are hyaluronic acid and gelatin, which can with the help of the release of free radicals from photoinitiators, initiated by UV light, polymerise, and form a hydrogel. In these hydrogels, cells can be encapsulated. The hydrogels can in turn be used to maintain cells as they are in the natural environment. For example, hydrogels can provide an in-vivo-like ECM for stem cells and endothelial cells by supporting “stemness” and cell-to-cell contact; respectively. We aim to establish a protocol for culturing cells in the hydrogelas a first milestone in a project focused on profiling the metabolome of cells grown in hydrogels. To accomplish this, four types of cells, namely mouse brain microvascular endothelial cells (bEnd.3), human umbilical vein endothelial cells (HUVECs), adult human lung fibroblast (hLFs) and mesenchymal stem cells (MSCs), were evaluated for growth in hyaluronic acid methacrylate (HA-ma), hyaluronic acid acrylamide (HA-am) as well as a QuattroGel composed by gelatin methacryloyl (GelMA), HA-ma, fibrinogen and thrombin. It was found that HA-masupported viability and the stemness of mesenchymal stem cells, of which the metabolome can be further studied in order to evaluate the difference between regular 2D maintenance and maintenance in 3D. No sprouting was observed for the other cells encapsulated in the hydrogel, and further experiments are needed to find the source of error.

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Electrospun nanofibers have been used for many applications, but a reliance on organic solvents limits their use in biomedical fields. In this study, we successfully electrospun nanofibers from aqueous chitosan-hyaluronic acid complex coacervates. We studied how solvent’ properties affected the average nanofiber diameter by using pure water as a solvent versus ethanol-water solutions. Experimentally, we investigated the effect of electrospinning apparatus parameters, such as how the applied voltage affected fiber formation and morphology. The smallest average nanofiber diameter was determined to be around 115 ± 30 nm when 3 wt% ethanol coacervate samples were electrospun using the applied voltage of 24 kV. Linear viscoelastic measurements were used to study the rheological characterization of complex coacervate with different salt concentrations and cosolvents (e.g., ethanol weight percent). Chitosan-hyaluronic acid nanofibers hold potential in biomedical applications such as wound dressing, tissue engineering, would healing scaffolds.

Despite the effort to optimize soft contact lens performance, almost half of the 140 million contact lens wearers worldwide experience symptoms of ocular dryness and discomfort, especially towards the end of the day. These symptoms are attributed to reduced compatibility between the contact lens and the ocular surface and are the main reason for contact lens discontinuation. As the interactions of the contact lens-eye interface are dynamic, the surface properties play a key role in improving ocular compatibility, comfort and overall performance of contact lenses. One promising method to reduce adverse interfacial interactions between the contact lens and the ocular surface is to modify the contact lens surface with a biomimetic layer inspired by the ocular surface and the tear film. Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan naturally found in the ocular environment providing ocular hydration and lubrication. Proteoglycan 4 (PRG4), a mucin-like glycoprotein naturally produced at the ocular surface contributes to natural lubrication during blinking and to tear film stability. Surface modification with HA or PRG4 has been shown to result in improved wetting, lubricating and antifouling properties. Moreover, HA and PRG4 have been previously found to interact and synergistically reduce friction further. In the current work, novel HA and PRG4-grafted soft contact lens surfaces were prepared, and the impact of the surface tethered layer on important contact lens properties was assessed. Furthermore, the potential synergistic effect between HA and rhPRG4 on the examined properties was evaluated. Surface immobilization of HA on model conventional (pHEMA) and silicone (pHEMA-co-TRIS) hydrogel contact lenses was achieved by thiol-ene “click” chemistry, while full-length recombinant human PRG4 (rhPRG4) was surface grafted via carbonyldiimidazole (CDI) linking chemistry respectively. The chemical structure after each modification step was determined by attenuated total reflectance FTIR (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analyses. HA-grafted model soft contact lenses were characterized by improved surface wettability, antifouling and water retentive properties, while a decreasing trend in boundary friction was observed but only for the HA-grafted pHEMA-co-TRIS materials. Surface-tethering of rhPRG4 was found to effectively enhance the surface wettability and boundary lubricating properties of pHEMA-co-TRIS hydrogels only, whereas both rhPRG4-grafted pHEMA and pHEMA-co-TRIS materials exhibited lower protein sorption and dehydration rate. Overall, the surface immobilization processes followed herein did not alter the optical transparency of the model soft contact lenses or their in vitro compatibility with human corneal epithelial cells. Finally, there was evidence that HA and rhPRG4 synergistically interacted, further improving the contact lens properties. However, the degree of HA/rhPRG4 synergy was found to be dependent on the configuration of the formed HA/rhPRG4 complex as well as the composition of the substrate hydrogel material, with the noted improvement being more significant for the model silicone hydrogels. This is the first study to examine surface grafted full-length rhPRG4 and the effect of this modification on contact lens properties. Moreover, the study is the first to investigate the interactions between covalently tethered rhPRG4 and solutions containing HA. The results of this thesis demonstrate that HA and rhPRG4 are good candidates for the development of novel biomimetic surfaces, especially for silicone hydrogel contact lenses. The potential for using these compounds in synergy was also demonstrated, with wetting solutions of HA showing promise for modifying rhPRG4 modified materials to improve symptoms of discomfort. These naturally occurring ocular agents have the potential to improve the management of ocular dryness and discomfort, thus optimizing the overall soft contact lens performance. / Thesis / Doctor of Philosophy (PhD)