published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The basic principle of tissue engineering is the combination of appropriate cells with biomaterials under conditions that promote and lead to tissue formation. A tissue engineering scaffold is a material that supports cells for their growth, proliferation, and differentiation in the absence of native extracellular matrix (ECM). The ECM was originally thought to provide primarily a mechanical support for the cells, but through receptors on the surface of cells, the ECM takes part in promoting cell adhesion, migration, growth, differentiation, and apoptosis. Ideally, a tissue engineered scaffold should mimic both the form and function of native ECM. Additionally, like any other biomaterial for implantation, a tissue engineered scaffold should be biocompatible and not initiate tissue reactions or immune responses. This work focuses on the evaluation of the biocompatibility of novel alloy-based materials for orthopedic applications. In addition, in the context of bone regeneration, it examines the influence of select native ECM constituents on mesenchymal stem cell (MSC) osteogenic differentiation in 3-D contexts. On the other hand, given the crucial role of vasculogenesis in cell nutrition in the scaffolds, ECM mimics found to support osteogenesis were further evaluated for endothelial cell adhesion and migration. For the hydrogel systems presented in this manuscript, poly(ethylene glycol) diacrylate (PEGDA) networks were selected as the base scaffold due to the broad tunability of their mechanical properties and their previous use in bone regeneration applications. In addition, pure PEGDA hydrogels do not intrinsically promote cell adhesion. Thus, cell interactions with PEGDA gels are initially isolated to the interactions supported by the proteins tethered to the scaffold. This work attempts to contribute to the development of novel materials that provide biocompatibility and enhanced versatility in orthopedic applications. Moreover, in the context of bone regeneration, the use of selective ECM biomolecules in hybrid hydrogel scaffolds will aid in the understanding of MSC osteogenic responses to specific ECM constituents. Additionally, incorporation of ECM mimics that support both osteogenesis and vasculogenesis will provide a more controlled platform which will serve as a foundation for the fabrication of more efficient vascularized bone constructs.

The soluble and readily extractable part of the plant extracellular matrix has been termed, the apoplast and contains a wide range of components such as, complex carbohydrates, structural proteins, enzymes and radicals that are known to be responsive to stress and developmental pressures. This thesis describes the development of a technique for the selective enrichment of apoplastic components for a range of subsequent analyses. Using this technique a number of apoplastic proteins were N-terminally sequenced and revealed 2 cell wall related enzymes, an antifungal protein and 3 auxin-binding/germin-like proteins. This technique also provided a novel approach to the further study of auxin-binding proteins via the use of affinity chromatography at their putative site of action, the apoplast. Three potential auxin-binding protiens were identified. Many attempts were made to subject the material extracted from the apoplast to the highly resolving technique of 2-dimensional electrophoresis, and during the process two unusual 2D systems were developed. These systems could be run in a small format that permitted very rapid analysis and/or using an in-gel loading strategy to subject up to 500µg of protein to 2D separation therefore permitting N-terminal sequencing from single 2D gels. Unfortunately 2D separation of apoplastic proteins was never fully achieved within the time frame of this study due to the vast degree of heterogenity present in the sample material. It did however demonstrate the very complex nature of apoplastic components. A series of experiments revealed that the tobacco leaf apoplast contained compartment specific antioxidant enzymes, some of which share physical characteristics with similar enzymes from other species. The activity of these enzymes altered in response to stress and according to the developmental age of the tissue. The reduced activity of these enzymes directly correlated to the degree of oxidative modification of apoplastic proteins illustrating that these enzymes are important in the detoxification of apoplastic radicals. Follow on experiments following the apoplastic generation of the superoxide anion and nitric oxide from impact stressed potato tuber tissue showed that radicals play important roles in the responses of plant tissue to stress, and show the first involvement of nitric oxide in plants in response to abiotic stress.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 292-320).

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaf 225-261).

Thesis (M.Orth.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 229-253). Also available in print.

Thesis (Ph. D.)--Illinois State University, 1992. / Title from title page screen, viewed January 27, 2006. Dissertation Committee: H. Tak Cheung (chair), Anthony Otsuka, Alan Katz, Brian Wilkinson, David Weber. Includes bibliographical references (leaves 108-120) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1989. / Title from title page screen, viewed October 12, 2005. Dissertation Committee: H. Tak Cheung (chair), David W. Borst, Herman E. Brockman, Arlan G. Richardson, Brian J. Wilkinson. Includes bibliographical references (leaves 145-156) and abstract. Also available in print.