University, Diss.--Helsinki, 1994.

Postoperative adhesions form as the body's natural response to injury in an effort to temporarily protect and supply nutrients to these tissues. However, adhesions can remain permanent, and fail otherwise successful surgeries by tethering tissues together that are normally separated. An ideal anti-adhesive device reduces unwanted adhesions and leaves the patient in a state most similar to before surgery. This dissertation introduces a novel, robust hydrogel film consisting of two hydrophilic polydisaccharides, hyaluronic acid (HA) and alginate. To address the challenge of retaining HA in alginate-rich hydrogels, we methacrylated the HA (GMHA), creating hydrophobic moieties. These hydrophobic interactions shift the percolation threshold, allowing for greater concentrations of GMHA to be retained in resulting films. UV crosslinking retains GMHA beyond the percolation threshold and widens the possibilities of usable films. To enhance the mechanical properties of these alginate/GMHA films, we employed a previously developed method for creating thin, branched, interconnected fibers using urea crystal templating. Templated films are softer and, yet, tougher than films that have not been templated. This toughness is a result of increased density of polymer in the fibers. These films were selected as most conformable and most robust by surgeons in a blinded handling study. In a rat peritoneal abrasion model for adhesion formation, the films successfully prevented adhesions with statistical equivalence to the leading anti-adhesion device commercially available. Finally, future recommendations are suggested for the development of a bilayer construct with a collagen/alginate blend bound to an alginate/HA layer for an anti-adhesive and regenerative strategy. This construct addresses the need for opposing strategies in the dynamic environment of wound healing. Further research is needed to develop the usefulness of this bilayer system, as preventing unwanted adhesions is merely a first step in achieving a blemish-free healed wound. / text

Laboratory analyses were conducted on synthetic iron oxides to assess the use of gamma (γ-) irradiation as an efficient sterilization technique to remove microorganisms present in natural bacteriogenic iron oxides (BIOS) and to determine if the technique induces mineralogical changes within the Fe-rich minerals. Fe-oxides (ferrihydrite, lepidocrocite, and goethite) were synthesized with and without alginate (as a proxy for exopolysaccharides) and microbial reductions were carried out using the bacterium Shewanella putrefaciens CN32. A total of 18 Fe-oxide minerals were subjected to microbial reduction to assess redox stability, alteration due to varying levels of gamma irradiation (0, 5, and 28 kGy), and the addition of the exopolysaccharide alginate. Iron reduction rates varied for each Fe-oxide with faster Fe (III) reduction rates observed for the amorphous poorly-sorted 2-line ferrihydrite and slower Fe (III) reduction for the more crystalline Fe-oxides lepidocrocite and goethite. There was no significant impact to the Fe (III) reduction rates due to gamma irradiation (p> 0.05), which was confirmed using a t-test for statistical variance between gamma irradiated samples. However, the addition of alginate enabled lepidocrocite and goethite to achieve maximum Fe (III) reduction by an average of 7 days faster when compared to the Fe-oxides synthesized without the exopolysaccharide.

No description available.

No description available.

Hemophilia A is an X-linked recessive bleeding disorder caused by the deficiency of coagulation factor VIII [1]. Current treatment for hemophilia A consists of prophylactic or on demand replacement therapy of either plasma-derived or recombinant FVIII concentrates [2]. Albeit effective, there are several limitations associated with factor concentrates, including high cost that limits its availability for close to 80% of hemophilia patients in developing countries [3-5]. An alternative treatment would thus be desirable. Gene therapy for hemophilia has seen many successes in animal models and represents a more cost-effective alternative to the current treatment modalities [6]. In the current work, I present a gene therapy system for hemophilia that uses mouse fetal myoblasts engineered to secrete FVIII, enclosed in immuno-protective alginate-poly-L-lysine-alginate (APA) microcapsules, as a sustained source of FVIII. In this study, a thorough examination of the encapsulated myoblasts using a novel flow cytometry assay was performed. This method yielded an accurate and precise method for encapsulated cell viability calculation, and also allowed for analysis of several other parameters such as health (cell morphology), cell size and distribution. Flow cytometry was also used to monitor the time-course proliferation profile of encapsulated myoblasts secreting cFVIII, using the division tracking dye CFSE. We found that encapsulated cells display a decreased proliferation rate as well as lower viability than non-encapsulated cells. Implantation of encapsulated G8 myoblasts secreting cFVIII into hemophilia A mice resulted in maximum plasma levels of protein on day 1 ( ~18% of normal canine FVIII levels). Delivery of cFVIII in hemophilic mice also offered protection against blood loss after the mice were subjected to injury (as measured by hematocrit levels); indicating that biologically functional cFVIII continued for at least 7 days post-capsule implantation. Low levels of FVIII antigen returned on day 28 after a transient disappearance on day 14. However, the presence of antigen must be reconciled with appearance of anti-cFVIII antibodies that were detected in the plasma of treated mice at the end of five weeks. The neutralizing nature of these antibodies still needs to be characterized by Bethesda assay Overall, our study demonstrates the feasibility of delivering therapeutic levels of FVIII using encapsulated G8 fetal myoblasts. The presence of functional FVIII protein on day 7, suggests that this treatment was not met by transcriptional repression in vivo, thereby overcoming one of the major obstacles faced by using the transformed C2C12 cell line secreting hFVIII [7] If such levels of FVIII were achieved in humans, it would be sufficient to convert a severe hemophiliac into a mild phenotype. Thus, this gene therapy strategy may be a suitable therapeutic alternative for hemophilia patients. Further work ought to focus on the long-term persistence of FVIII in hemophilia A mice, and also determining the protection following trauma over time to determine if the FVIII remains functional. Other cell lines should be explored for higher expression, reduced immunogenicity and improved viability Still, there is a need to develop human cells expressing high levels of biologically active hFVIII with similar properties to the fetal cells described in this study [8, 9] / Thesis / Master of Applied Science (MASc)

A biosensor is proposed that contains bacteria that naturally effluxes potassium ions when threatened by electrophilic species. Pseudomonas aeruginosa is an activated sludge isolate and possesses the characteristic potassium efflux response. It has been immobilized in calcium alginate beads, photopolymer disks, and a thermally reversible gel in order to ultimately incorporate the immobilized system into a functional biosensor. The potassium efflux and cell viability were measured in the immobilized matrices. Wastewater treatment is of utmost importance; however, processes are easily upset. Upsets can be caused by various electrophiles found in the environment, and can cause serious health effects to people or the environment downstream from an upset. Electrophiles can cause the activated sludge in wastewater treatment facilities to deflocculate, and untreated water can be lost downstream. Devising a detection system for proactively sensing electrophiles prior to an upset is an important complementary goal. Immobilization systems have been evaluated including photopolymer coated alginate beads and sol gel coated alginate beads. The thermally reversible gel, NIPA-co-AAc (N-isopropylacrylamide-co-acrylic acid), shows promise as an immobilization matrix for the bacteria; however its high lower critical solution temperature (LCST) of ~33oC is problematic for typical, ambient applications. Another thermally reversible copolymer, N-isopropylacrylamide-co-N-acryloyl-6-amino caproic acid (NIPA-co-AcACA) was synthesized; however, it did not form a continuous matrix; making it useless as an immobilization scheme for biosensors. Alginate beads fall apart easily in bacteria media, but are structurally stable in potassium solutions. Cells immobilized in alginate beads seemed to efflux four times less potassium than did planktonic controls, while cells in thermally reversible gels effluxed a comparable amount of potassium as planktonic controls. This result may indicate a tighter matrix around the alginate immobilized cells, not allowing proper diffusion of potassium out of the matrix. / Master of Science

<p> Cell encapsulation aims at the delivery of a therapeutic protein to a patient from transplanted cells. Conventional approaches involve immune-isolating cell lines that have been genetically modified to express a therapeutic protein, in alginate-based microcapsules. The long-term success of this approach hinges on the structural stability of the microcapsules, as well as their ability to maintain an environment suitable for the long-term survival of encapsulated cells. The most commonly studied type of microcapsule is the alginate-poly-Llysine-alginate (APA) microcapsule. However, the main concern with AP A microcapsules is the Joss of structural integrity during long-term implantation due to the exchange of calcium ions with other physiological ions, as well as the loss of the polyelectrolyte overcoats. </p> <p> In order to increase the structural stability of the microcapsules, we developed and characterized a number of synthetic polyelectrolytes that undergo phase separation upon complexation, and which are capable of forming covalent cross-links. These reactive polyelectrolytes are designed to take the place of poly-L-lysine and the outer alginate layer. We also explored combining cross-linkable synthetic polyanions with sodium alginate to strengthen the Ca Alginate core, by forming a core cross-linked network extending throughout the microcapsules. The polyelectrolyte complexes, encapsulation processes and microcapsule properties were studied in detail using extensive characterization techniques, including collaborative work on cell viability and host-immune response. </p> <p> Overall, this thesis describes a novel approach and prom1smg materials for cell encapsulations that offer enhanced microcapsule resistance to chemical and mechanical stresses, while preserving the desired biocompatibility. These materials may ultimately be useful for clinical immunosuppressive therapies. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

The opportunistic pathogen P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. During chronic infection of the cystic fibrosis lung, P. aeruginosa undergoes conversion to a mucoid phenotype, constitutively producing the exopolysaccharide alginate, composed of the uronic acids D-mannuronate and L-guluronate. This alginate production contributes significantly to virulence in the cystic fibrosis lung. Evidence suggests that the acetylation state of the mannuronate component of the alginate influences the ability of components of the immune system to phagocytose the organism. To garner new and relevant information regarding the mechanism of alginate acetylation in Pseudomonas aeruginosa, a variety of approaches were undertaken. Analysis of the alginate produced by algX, algG, and algK alginate biosynthesis mutants revealed that the small oligouronides they produced were unacetylated. This strongly supports the hypothesis that the mannuronates are acetylated in periplasm, and that a polymer of at least some specific size is required. While three alginate biosynthesis gene products (AlgI, AlgJ, and AlgF) have been shown to be involved in alginate acetylation, another gene in the cluster, algX, shares 30% identity with one of them and thus generates speculation as to its potential involvement in the process. To test this possibility, an algX mutant was complemented with a plasmid carrying a mutation at a conserved residue shown to be required for alginate acetylation in the homologous protein. Analysis of alginate from this construct suggested that AlgX is not involved in alginate acetylation. To determine if changes in levels of alginate acetylation are accomplished at the transcriptional level, transcript levels of several alginate biosynthesis genes in different media were determined by real-time PCR. As qRT-PCR had not been previously performed on any of the alginate biosynthesis genes, this yielded important information about the transcription of the operon. In addition, beta-galactosidase assays on upstream regions of several biosynthesis genes identified two previously unrecognized promoters, one upstream of algG and one upstream of algI. The remaining approach was to examine protein interactions of AlgF, the protein product of one of the three acetylation genes. 2-D redox SDS-PAGE gels indicated that disulfide bonding may be important for interactions with this protein. While mass spectrometry was unable to identify the binding partners of AlgF, efforts are ongoing to create a mutation in the P. aeruginosa genome that changes the cysteine residue in AlgF to a serine residue. This would be a definitive method for determining the importance of disulfide bonding in AlgF.