Cells respond to chemical, mechanical and topographical cues both in vivo and in vitro. Much research has been carried out into the effects of chemical signals and to a lesser degree, mechanical. However, less is known about cell responses to topographical cues, particularly to topographies with nanoscale dimensions. Understanding how cells respond to topography is of particular interest to the field of tissue engineering, where it is crucial to characterise the effects that biomaterial surfaces have on the cells that they come into contact with. Observations of the impact that topographic signalling has on cells, within two tissue engineering systems, are discussed in this thesis. These systems are: polymer conduits for peripheral nerve regeneration and thin films for the replacement of the retinal pigment epithelium. Understanding the effects that micro and nano scaled topographies have on nerve and retinal cell regeneration is important for successful development and implementation of appropriate tissue engineered devices. In order to fabricate topographical patterns on biomaterial surfaces, a number of fabrication techniques were investigated. The fundamental requirement of these techniques was for reliable production of uniform nano and micro scale topographical patterns over large lateral areas (millimeter scale). Initially, the suitability of electrohydrodynamic lithography (EHDL) was assessed. EHDL is a relatively new technique, first published in 2000, which employs electrostatic forces to pattern thin polymer films. Subsequently, techniques traditionally associated with the computing industry, such as e-beam lithography and reactive ion etching, were evaluated. Following successful pattern fabrication, NG108-15 and ARPE-19 cells were cultured on grooved topographies. Against a baseline parameter of elapsed time, the cell morphologies and their propensity for alignment with the grooves was rigorously assessed and compared. ARPE-19 and NG108-15 cell responses differed from one another, and were sensitive to varying groove dimensions. Ultimately, the developing morphologies (for both cell types) proved to be clearly dependent on groove dimensions and elapsed time.

A 7-9 fold enriched preparation of bovine retinal pigment epithelial cell plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight retinal pigment epithelial cellular adhesion to the rod photoreceptor cells. The contaminating rhodopsin was partially removed by anti-rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or silver. Monoclonal antibodies raised against the retinal pigment epithelial plasma membrane preparation cross reacted with rod outer segment preparations. A monoclonal antibody, designated Rho-5A3, was classified as an IgG₃ kappa light chain immunoglobulin. It was shown to be specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment membrane proteins electrophoretically transferred to CNBr-activated paper. Limited proteolytic digestion of rhodopsin followed by electrophoretic transfer to CNBr- activated paper localized the binding site of this antibody to the N-terminal two-thirds of the rhodopsin molecule. Competition assays with rhodopsin polypeptides further defined the antigenic site to be within the 17-39 amino-acid segment of rhodopsin. The Rho-5A3 antibody did not bind to sealed ROS discs or frozen-thawed ROS discs but did bind to Triton X-100 solubilised discs indicating a detergent solubilisation dependence for antigenic site accessibility. Cultures of bovine retinal pigment epithelial cells were started by initial enzymatic isolation followed by recovery in RPMI-1640 culture medium. The retinal pigment epithelial cells established a doubling time of 52 hours until the cells reached culture confluency . The cells also maintained many of their in vivo characteristics such as a high degree of pigmentation and an abundance of microvilli. Cell surface glycoproteins labelled with FITC-Con A, FITC-WGA, and FITC-RCA showed dense and random surface labelling patterns. Fluorescent labels were induced to redistribute to central spots and clear from the cell surface by incubating the labelled cells in buffer for 60 minutes at 37°C. Treatment by the appropriate saccharide inhibitors indicated that the labelled sites had undergone endocytosis by the cell. Continuous labelling experiments indicated that redistribution and internalization is constantly occurring so that previously unlabeled receptors become accessible for labelling. As a result a dense pattern of label on the cell surface was maintained. The protein actin, with apparent Mr =46,000, was detected with rabbit anti-actin antisera labelling of the RPE plasma membrane preparation proteins electrophoretically transferred to nitrocellulose paper. Immunofluorescent labelling using the rabbit anti-actin antisera confirmed biochemical studies that actin was a major component of the bovine RPE cell. The activation of actin filaments may play an important role in the phagocytosis of bovine rod outer segments by retinal pigment epithelial cells in tissue culture. Scanning electron microscopy and transmission electron microscopy have shown that 2 week old bovine retinal pigment epithelial cells in vitro can be induced to recognize, attach, and engulf dark adapted sealed rod outer segments. In summary a monoclonal antibody was raised against a bovine retinal pigment epithelial cell plasma membrane preparation, however, the antibody raised proved to be specific for rhodopsin, a contaminating ROS protein found in the preparation. The monoclonal antibody, designated Rho-5A3, was fully characterized and its antigenic site determined. Finally, bovine retinal pigment epithelial cells grown in tissue culture acted as a model system for studying cell surface components and rod outer segment phagocytosis at the levels of fluorescence, scanning electron microscopy, and transmission electron microscopy. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This investigation had for its aim the determination of the effects of various materials, added to the basic culture medium, upon pigment production of the four chromogens Staphylococcus aureus, Sarcina lutea, Serratia marcescens, and Rhodococcus cinnabaris.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

The problem with which this study was concerned was that of developing wood stain formulas from a small supply of materials. These formulas should produce a wide variety of colors from which to select, the use of which should be suited to the school or home laboratory.