Heart failure is an important medical disease and impacts millions of people throughout the world. In order to treat this problem, biomaterial injectable treatment injected into the myocardium of the failing LV are currently being developed. Through this treatment, the biomaterial material injections can reduce wall stresses during the cardiac remodeling process. By using computational techniques to analyze the effects of a treatment involving the injection of biomaterial material into the LV after MI, the material parameters of the hydrogel injections can be optimized. The results shows that the hydrogel injections could reduce the global average fiber stress and the transmural average stress seen from optimization. These results indicated that the hydrogel injections could influence the stiffness in passive LV tissue, but there is still need for more research on the active part of ventricular contraction. Conclusion: hydrogel injection is a viable way to alter ventricular mechanical properties.

Baculoviruses are versatile viruses that can be used as biopestisides, or for the production of recombinant protein and vaccines. Baculoviruses have also been found to be able to transfer genes to mammalian cells. This finding opened the door for the application of baculovirus vectors in human gene therapy. However, the mass production of clinical grade baculovirus vectors is challenging. Downstream processing has now become the bottle-neck of the manufacturing process. In this work, an anion exchange chromatography-based process was investigated for the purification of recombinant baculovirus vectors using a novel hydrogel based membrane (Natrix Separations Ltd.). Crude recombinant baculovirus supernatant from infected insect cell cultures was first subjected to a clarification process consisting of centrifugation and filtration. The pH of the viral solution was adjusted and then passed through a fast protein liquid chromatography system consisting of the ion exchange membrane. After washing weakly bound impurities, the captured baculoviruses are recovered by an elution step. Overall, baculoviruses strongly associated with the membrane; however, this interaction which was much physical as it was chemical, could not be entirely reversed and baculovirus was lost in the process. Product purity has also been evaluated and up to 85% of total protein reduction was determined. The significant losses of baculovirus observed have indicated major limitations in using this membrane for the purification of baculovirus.

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the embryo that have great potential for regenerative therapies because of their ability to self-renew and differentiate into almost all cell types. However, this developmental potential is influenced by the local cellular microenvironment, including cell surface bound ligands. In this study, we synthesized an artificial stem cell niche wherein vascular endothelial growth factor A (VEGFA) was functionally immobilized in an agarose hydrogel. Immobilized VEGFA treatments were able to upregulate mesodermal markers, brachyury and VEGF receptor 2, by day 4 and were CD34+CD41+ by day seven. Subsequently, VEGFA immobilized treatments were able to generate colony forming cells by day fourteen. This work demonstrates our ability to use functionalized hydrogels to guide ESCs toward blood progenitor cells and serves as a useful tool to replicate aspects of the embryonic microenvironment.

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the embryo that have great potential for regenerative therapies because of their ability to self-renew and differentiate into almost all cell types. However, this developmental potential is influenced by the local cellular microenvironment, including cell surface bound ligands. In this study, we synthesized an artificial stem cell niche wherein vascular endothelial growth factor A (VEGFA) was functionally immobilized in an agarose hydrogel. Immobilized VEGFA treatments were able to upregulate mesodermal markers, brachyury and VEGF receptor 2, by day 4 and were CD34+CD41+ by day seven. Subsequently, VEGFA immobilized treatments were able to generate colony forming cells by day fourteen. This work demonstrates our ability to use functionalized hydrogels to guide ESCs toward blood progenitor cells and serves as a useful tool to replicate aspects of the embryonic microenvironment.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

ABSTRACT HYDROGEL FROM TEMPLATE POLYMERIZATION OF METHACRYLIC ACID AND N-VINYLPYRROLIDONE AND POLYETHYLENEOXIDE Yelda, Erdem Department of Polymer Science and Technology Supervisor : Prof. Dr. Teoman Tin&ccedil / er April 2005, 53 pages This theses covers the preparation and the characterization of a rigid hydrogel from N-Vinyl pyrrolidone-methacrylic acid (VP-MAA) monomers and polyethyleneoxide (PEO) polymer. Hydrogels are hydrophillic natured three dimensional networks which can swell in the presence of water. The VP-MAA-PEO hydrogel was obtained by template polymerization which can be defined as a method of polymer synthesis in which specific interactions consists of the preparation of a polymer (daughter polymer) in the presence of a macromolecular compound (template polymer). The hydrogel of VP-MAA-PEO was synthesized by using azobisisobutyronitrile (AIBN) as the initiator, tetrahydrofurane (THF) as the solvent and the temperature of the system was kept constant at 50&ordm / C. Two kinds of VP-MAA-PEO hydrogels were prepared. The only difference between them were their solubilities in water. This difference is due to different crosslinking agent weight percentages of ethylene glycol dimethacrylic (EGDMA) to make the hydrogel water insoluble. The comparison of two hydrogels were carried by swelling behaviors at different pH values and different temperatures. Thermal stability of these two hydrogels were also examined by differential scanning calorimetry (DSC), spectroscopic properties were compared by using FTIR spectrometer and morphological studies were analyzed by using scanning electron microscope (SEM).

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23921号 / 工博第5008号 / 新制||工||1782(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 古賀 毅, 教授 中村 洋, 教授 竹中 幹人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Chronic ulcers are an important and costly medical issue, causing their sufferers a large amount of pain, immobility and decreased quality of life. The common pathology in these chronic wounds is often characterised by excessive proteolytic activity, leading to the degradation of both the extracellular matrix, as well as key factors critical to the ulcer's ability to heal. As matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), it was hypothesised that this specific proteolytic activity was directly related to an ulcer's chronic nature. Although previous studies have identified elevated proteases in CWF, many have reported contradictory results and therefore the precise levels and species of MMPs in CWF are poorly understood. The studies reported herein demonstrate that MMP activity is significantly elevated in CWF compared with acute wound fluid (AWF). In particular, these studies demonstrate that this proteolytic activity can be specifically attributed to MMPs and not another class of proteases present in wound healing. Furthermore, it is shown that MMP-9 is the predominant protease responsible for matrix degradation by CWF and is an indicator of the clinical status of the wound itself. Moreover, MMP-9 can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and also tethered to a synthetic biocompatible hydrogel compromised of aqueous poly (2-hydroxy methacrylate) PHEMA synthesised in the presence of poly(ethylene glycol) (PEG). Together, these results highlight the potential use of a tethered MMP inhibitor as an improved ulcer treatment to inhibit protease activity in the wound fluid, while still allowing MMPs to remain active in the wound bed where they perform vital roles in the activation of growth-promoting agents and immune system regulation.

Techn. Universiẗat, Diss., 2006--Darmstadt.