The investigation of surface chemical and nanotopographical cues to engineer biointerfaces
This thesis is focused on understanding the fundamental physical interaction occurring, when a material interacts with a biological fluid containing protein molecules and cells. The interaction of proteins with defined surface chemistry and nanotopography is of major interest in the field of biomaterials. Despite the degree of research undertaken to study this interface, there is still a lack of understanding of how the protein layer evolves with respect to surface parameters, and further how cells condition the surface. By understanding these processes, advanced biomaterial coatings may be developed that allow control over specific cell responses to direct healing processes.
Colourimetric and fluorometric assays were carried out to assess protein-surface affinity and amount of protein adsorption. Infrared spectroscopy was used to quantify protein conformational changes incurred upon adsorption on defined nanoscale surfaces presenting a range of chemical functional groups. Model experiments were performed using bovine albumin and fibrinogen adsorbing onto surfaces presenting defined surface chemistry: OH, COOH, NH2, and CH3. Surface curvature on the nanoscale was used to model topography on the same length scale as protein molecules. Silica colloidal dispersions were prepared in batches,11-215 nm diameters allowing chemical modification whilst keeping nanotopography constant.
3T3 fibroblasts were cultured over a library of surfaces presenting a spectrum of batches chemical functionality and nanostructure. Changes in cell attachment, morphology, migration and proliferation were examined. Media was removed at two different time points of 30 minutes and 24 hours, and examined to identify changes in fibroblast secreted proteins. Liquid chromatography was used to separate the cell culture media after incubation with cells over various chemically functionalised surfaces. Electrospray ionisation (ESI) and matrix
assisted laser desorption (MALDI) mass spectrometry were used to identify changes in media with respect to the varying surfaces used and over time.
The studies presented in this thesis give a better understanding of the interaction between silica nanoparticles and protein molecules, including conformational changes that occur when protein adsorbs on the nanoparticle; the effect of surface nanotopography and defined chemistry on protein adsorption is examined with respect to both chemical functionality and nanotopography. Clear differences were observed in the amount of protein adsorbed and its structural presentation when bound. The strength of the interaction, described through isotherm fitting, gave insight into the mechanism of competitive protein binding. Surface curvature on the nanoscale was also found to act synergistically with surface chemistry to dictate the dynamic accumulation of protein at the surface interface. In the later chapter discussion is given in terms of cell-surface interaction. Experimental evidence is shown for different mass spectroscopic analysis of reduced complexity media following initial cell-surface interaction and that after 24 hours. From this it is postulated that cell secretions are effected through interaction with the surface, with these changes being significant even after 30 minutes of cell culture with the defined surfaces. These changes are specific to the presented surface as they do not alter with respect to longer culture periods, but media are clearly different collected from cells cultured on different surfaces.
This research will help solve challenges facing materials science, understand biological responses to surroundings and help in the development and advance of medical devices, drug delivery, therapeutics and diagnostics.
|Jan 1, 2015