Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system and are thought to be responsible for the successful directional growth of new olfactory axons throughout the life of adult mammals. Due to this unique property, OECs have been targeted as a potential cellular transplantation therapy for spinal cord injury. In order to effectively isolate OECs for intraspinal transplantation, more knowledge must be gained on their phenotypic properties. We investigated the neurochemical features of OECs in a variety of mammalian species (including hamsters, rabbits, monkeys, mice, and pigs) using three biomarkers: glial fibrillary acidic protein (GFAP), S100β, and α-smooth muscle actin (αSMA). In addition, we tested the ability of a few biomatrices to sustain and promote OEC growth and survival in vitro. The rationale for using biomatrices is to provide a supportive environment for glial and axonal growth in the spinal lesion. Here, we found that mucosal and bulbar OECs from all five of the aforementioned mammalian species express S100β. Expression of GFAP, however, was not consistent across the five species. Both mucosal and bulbar OECs of monkeys express αSMA; only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Though αSMA immunostaining was not detected in the OECs of adult mice, in adult mutant mice lacking αSMA expression, OECs displayed perturbed ultrastructural morphology. None of the biomatrices used (methacrylated glycol chitosan, arginine-glycine-aspartic acid – grafted methacrylated glycol chitosan, and agarose) were able to promote OEC proliferation. Isolated strips of rodent olfactory lamina propria (the deep connective tissue layer in the olfactory mucosa containing primary sensory axons and OECs) showed sustained growth when cultured for 10 days. In sum, these findings highlight the following points: the efficacy of S100β and αSMA as biomarkers for mammalian OECs in vivo; the potential for isolated strips of lamina propria to provide a natural, supportive environment for OECs during intraspinal transplantation; the failure of methacrylated glycol chitosan and its derivatives, as well as agarose, to promote OEC proliferation. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-07-27 15:29:47.642

In this work, the development of temperature- and protein-responsive sensor materials based on biocompatible, inverse hydrogel opals (IHOs) is presented. With these materials, large biomolecules can be specifically recognised and the binding event visualised. The preparation of the IHOs was performed with a template process, for which monodisperse silica particles were vertically deposited onto glass slides as the first step. The obtained colloidal crystals with a thickness of 5 μm displayed opalescent reflections because of the uniform alignment of the colloids. As a second step, the template was embedded in a matrix consisting of biocompatible, thermoresponsive hydrogels. The comonomers were selected from the family of oligo(ethylene glycol)methacrylates. The monomer solution was injected into a polymerisation mould, which contained the colloidal crystals as a template. The space in-between the template particles was filled with the monomer solution and the hydrogel was cured via UV-polymerisation. The particles were chemically etched, which resulted in a porous inner structure. The uniform alignment of the pores and therefore the opalescent reflection were maintained, so these system were denoted as inverse hydrogel opals. A pore diameter of several hundred nanometres as well as interconnections between the pores should facilitate a diffusion of bigger (bio)molecules, which was always a challenge in the presented systems until now. The copolymer composition was chosen to result in a hydrogel collapse over 35 °C. All hydrogels showed pronounced swelling in water below the critical temperature. The incorporation of a reactive monomer with hydroxyl groups ensured a potential coupling group for the introduction of recognition units for analytes, e.g. proteins. As a test system, biotin as a recognition unit for avidin was coupled to the IHO via polymer-analogous Steglich esterification. The amount of accessible biotin was quantified with a colorimetric binding assay. When avidin was added to the biotinylated IHO, the wavelength of the opalescent reflection was significantly shifted and therefore the binding event was visualised. This effect is based on the change in swelling behaviour of the hydrogel after binding of the hydrophilic avidin, which is amplified by the thermoresponsive nature of the hydrogel. A swelling or shrinking of the pores induces a change in distance of the crystal planes, which are responsible for the colour of the reflection. With these findings, the possibility of creating sensor materials or additional biomolecules in the size range of avidin is given. / In dieser Arbeit wird die Entwicklung von temperatur- und proteinresponsiven Sensormaterialien auf Basis von biokompatiblen, inversen Hydrogelopalen (IHO) vorgestellt, mit welchen die spezifische Erkennung größerer Biomoleküle visuell ausgelesen werden kann. Die Darstellung der IHOs erfolgte mittels Templatverfahren, bei dem im ersten Schritt monodisperse Silicapartikel vertikal auf Objektträger abgeschieden wurden. Die so erhaltenen Kolloidkristalle mit einer Dicke von 5 μm zeigten opaleszente Reflexionen aufgrund der gleichförmigen Anordnung der Partikel. Im zweiten Schritt wurde das Templat in eine Matrix aus biokompatiblen, thermoresponsiven Hydrogelen eingebettet. Die Comonomere wurden aus der Familie der Oligo(ethylenglykol)methacrylate ausgewählt. Zur Synthese des Hydrogels wurde die Monomerlösung in eine Polymerisationsform injiziert, welche die Kolloidkristalle als Templat beinhaltete. Die Zwischenräume der Templatpartikel wurden mit der Monomerlösung gefüllt und das Hydrogelnetzwerk per UV-Polymerisation erhalten. Die Templatpartikel wurden anschließend nasschemisch heraus gelöst, so dass eine poröse innere Struktur erhalten wurde. Die regelmäßige Anordnung der Poren und damit die opaleszenten Reflexionen wurden dabei beibehalten, so dass diese Systeme als inverse Hydorgelopale bezeichnet werden. Ein Porendurchmesser von mehreren hundert Nanometer, sowie durchgängige Verbindungskanäle zwischen den einzelnen Poren sollten eine Diffusion von großen (Bio)molekülen erleichtern, was bei bisherigen Systemen ein Problem darstellte. Die Copolymerzusammensetzung wurde dabei so gewählt, dass ein Kollaps des Hydrogels über 35 °C stattfand. Alle Hydrogele zeigten ausgeprägte Quellung in Wasser unterhalb der kritischen Temperatur. Der Einbau von reaktiven Comonomeren mit Hydroxylgruppen gewährleistete dabei die Funktionalisierbarkeit des Hydrogels mit Erkennungsgruppen für entsprechende Analytmoleküle, wie z.B. Proteine. Als Testsystem wurde Biotin als Erkennungseinheit für Avidin in das Hydrogel mittels polymeranaloger Steglich Veresterung eingebaut. Die Menge an zugänglichem Biotin wurde dabei per colorimetrischem Bindungsassay quantifiziert. Dabei zeigte sich, dass sich die Wellenlänge der Reflexion nach Zugabe von Avidin zum biotinylierten inversen Hydrogelopal signifikant verschob und damit das Bindungsereignis visuell auslesbar ist. Dieser Effekt beruht auf dem veränderten Quellungsverhalten des Hydrogels nach Bindung des hydrophilen Proteins Avidin in Wasser, welches durch den thermosresponsiven Charakter des Hydrogels verstärkt ist. Ein Aufweiten oder Schrumpfen der Poren ändert die Abstände der gleichmäßig angeordneten Poren, welche für die Farbe des inversen Opals verantwortlich sind. Auf Basis dieser Erkenntnisse lassen sich möglicherweise Sensormaterialen für die Erkennung weiterer Biomoleküle in der Größenordnung von Avidin erstellen.

Hydrogel materials have become a heavily studied as materials for interfacing with biology both for laboratory investigations and the development of devices for biomedical applications. These polymers are water swellable and can be made responsive to many different stimuli by choice of monomers, co-monomers, and cross-linkers or functionalization with pendent ligands, substrates, or charged groups. The high water content, low moduli and potential responsively of these polymers make good candidates for biomaterials. A specific type of hydrogel called a microgel or a hydrogel micro/nanoparticle has similar properties to bulk hydrogel materials. Many of the interesting results and utility of the microgels in bioapplications are due to their inherent softness of the material. Here, the softness, flexibility, and conformability of these water swollen particles is used to create an interesting sensor platform, studied in the context of a microgel passing through a pore, and used as an emulsifier to create a drug delivery platform. The unifying theme of this dissertation is the softness of microgels which is critical for all of these experiments. However, the study of individual microgel softness is challenging and complex, since the softness is composed of two different components. The first is that the microgel is a swollen polymer which can be deswollen by an external stimuli or force. The second is that the microgel is a volume conserving elastic colloid which can deform without deswelling under the certain conditions. Throughout, this dissertation will discuss the ramifications of the complex softness of microgels in each experimental result and potential application.

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.

Nowadays, cancer treatment and tissue regeneration have attracted large amount of attention. Single Wall Carbon Nanotubes (SWNT) possess large surface area and outstanding optical and electrical performance, making it a promising component in cancer therapy and tissue reengineering systems. In this study, four disease treating systems based on SWNT are developed. They are pH-sensitive poly(ethylene glycol)-doxorubicin(PEG-DOX)@SWNT drug release system, temperature sensitive SWNT hydrogel, SWNT based biocompatible magnetic hydrogel and biocompatible SWNT-gelatin-F127-cysteamine hydrogel for tissue engineering. The successfully synthesized target compounds are characterized by FTIR. The in vitro release of drugs from the drug release systems is evaluated upon changes of pH values and the laser scanning. The effect of cancer treatment systems on specific kind of cells are examined by confocal laser scanning microscopy (CLSM). The results indicate that all of the four systems show great potential in the biomedical applications especially in disease therapy applications.

Microgels are lightly cross-linked hydrogel particles in the sub-micrometer to micrometer size range with a capacity to drastically change their volume in response to changes in the external environment. Microgels have an ability to bind and store substances such as biomacromolecular drugs, notably proteins and peptides, and release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials. This thesis aims at clarifying important factors affecting peptide-microgel interactions. These interactions were studied by micromanipulator-assisted light and fluorescence microscopy focusing on microgel deswelling in response to peptide binding, as well as re-swelling in response to peptide release or enzymatic degradation. To evaluate peptide uptake in microgels, solution depletion measurements were used whereas the peptide secondary structure was investigated by circular dichroism. In addition, the peptide and enzyme distribution within microgels was analyzed with confocal microscopy. Results presented in this thesis demonstrate that peptide incorporation into microgels, as well as peptide-induced microgel deswelling, increases with peptide length and charge density. In addition, results demonstrate that the peptide charge (length) rather than peptide charge density determines microgels deswelling. End-to-end cyclization is shown to not noticeably influence peptide-microgel interactions, suggesting that peptide cyclization can be used in combination with oppositely charged microgel carriers to improve the proteolytic and chemical stability of the peptide compared to the corresponding linear variant. Peptide secondary structure is found to drastically affect peptide incorporation into, and release from, oppositely charged microgels. Furthermore, it is shown that microgel charge density, peptide molecular weight, and enzyme concentration all greatly influence microgel bound peptide degradation. Of importance for applications, protective effects of microgels against proteolytic peptide degradation are observed only at sufficiently high microgel charge densities. Enzyme-mediated microgel degradation is shown to increase with increasing enzyme concentration, while an increased peptide loading in microgels causes a concentration-dependent decrease in microgel degradation. Taken together, results obtained in this work have provided some insight into factors of importance for rational use of microgels as delivery systems for protein or peptide drugs, but also in a host of other biomedical applications using weakly cross-linked polymer systems.

Due to an increased environmental awareness and thus, concerns over the use of fossil-based monomer for polymer production, there is an ongoing effort to find alternatives to non-renewable traditional monomers. This has ushered in the rapid growth in the development of bio-based materials such as green monomers and biodegradable polymers from vegetable and animal resources. Glycerol, as a renewable bio-based monomer, is an interesting candidate for sustainable polymer production. Glycerol is a renewable material that is a by-product of the transesterification of vegetable oils to biodiesel. Utilization of the excess glycerol derived from the growing biodiesel industry is important to oleochemical industries. The main objective of this thesis was to produce high molecular weight polyglycerol from glycerol and synthesize stimuli-responsive polyglycerol hydrogels. The work began with an investigation of the step-growth polymerization of glycerol to relatively high molecular weight polyglycerol using several catalysts. The catalytic reaction mechanisms were compared and the polymer products were fully analyzed. High molecular weight partially branched polyglycerol with multimodal molecular weight distributions was obtained. The polymerization of glycerol proceeded fastest with sulphuric acid as catalyst as indicated by the highest observed conversion of monomer along with the highest molecular weights. Theoretical models were used to predict the gel point and to calculate monomer functionality. High molecular weight polyglycerol was used to synthesize novel stimuli-responsive hydrogels. Real-time monitoring of step-growth polymerization of glycerol was investigated using in-line and off-line Attenuated Total Reflectance/Fourier Transform infrared (ATR-FTIR) technique.

Currently, organ transplant procedures are insufficient to address the needs of the number of patients that suffer of organ failure related disease. In the United States alone, only around 19% of the patients are able to get an organ transplant surgery and 25% die while waiting for a suitable donor. Tissue engineering (TE) has emerged as an alternative to organ transplant; thus, the aim of the present study was to validate a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel system as a model for material scaffolding in TE applications. This work explores the influence of scaffold material properties on cell behavior. Specifically, scaffold modulus, mesh size, and biochemical stimuli were characterized and their influence on cell response was analyzed at the biochemical, histological and microenvironmental levels. Three different TE targets were evaluated: vocal fold restoration, vascular grafts and osteochondral applications. Vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by initial scaffold mesh size and modulus. The results showed increasing levels of SM-α-actin and collagen production with decreasing initial mesh size/increasing initial modulus, which indicated that VFFs were induced to take an undesirable myofibroblast-like phenotype. In addition, it was possible to preserve VFF phenotype in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). On the other hand, regarding vascular graft applications, smooth muscle cell (SMC) phenotype was enhanced by increasing scaffold mesh size and modulus. Finally, the effect of scaffold inorganic content (siloxane) on rat osteoblasts and mouse mesenchymal stem cells was evaluated. Interestingly, the impact of inorganic content on cell differentiation seemed to be highly dependent on the initial cell state. Specifically, mature osteoblasts underwent transdifferentiation into chondrocyte-like cells with increasing inorganic content. However, Mesenchymal stem cells appeared to be preferentially driven toward osteoblast-like cells with an associated increase in osteocalcin and collagen type I production.

Stuttgart, Univ., Diss., 2002.

Universiẗat, Diss., 2001--Heidelberg.