The overall thrust of this dissertation is to gain a fundamental understanding of the synergistic effects between surface topography and chemical functionality of poorly adhesive materials on enhancing the adhesion of mouse embryonic fibroblasts. Cellular response to surface topography and chemical functionality have been extensively studied on their own providing valuable information that helps in the design of new and improved biomaterials for tissue engineering applications. However, there is a lack of understanding of the synergistic effect of microscale and nanoscale topography with chemical functionality and the relative impact and contribution of each in modulating cellular behavior. By understanding the relationship between these cues, in particular using materials that are poorly adhesive, this study will provide new clues as to how cells adapt to their environment and also suggest new dimensions of biomaterial design for fine-tuning cellular control. A microstructure that combined non adhesive materials with defined surface topography and surface chemistry is presented, to assess and correlate the enhancement of mouse embryonic fibroblasts cell adhesion and spreading. Poly (N-isopropylacrylamide) or PNIPAAm electrospun fibers were overlaid on PNIPAAm thin films (100 nm) at various time points to investigate the role of topography on such coatings by keeping the chemical functionality the same. After doing this, several topographical patterns were developed, spanning from sparse to dense fiber mats, and cell adhesion strongly depended on the relative available areas for attachment on either the fibers or the supporting surface. To gain a better understanding of this finding, two surface chemistries, non-adhesive (self-assembled monolayer of polyethylene glycol (PEGSAM) alkanethiol on gold) or an adhesive coating (3-aminopropyltriethoxysilane (APTES) on glass) with well characterized adhesive properties were included in this study to assess the effect of topographical cues provided by the PNIPAAm electrospun fibers on cellular responses. With the deposition of the PNIPAAm fibers onto a PEGSAM surface, cell adhesion increased to almost 100%, and unlike the PNIPAAm surface, cell spreading was significantly enhanced. With the deposition of PNIPAAm fibers onto APTES, both cell adhesion and spreading were unaffected up to 60% fiber coverage. For both surfaces, PNIPAAm fiber densities above 60% coverage lead to adhesion and spreading independent of the underlying surface. These findings indicate the presence of a sparse topographical feature can stimulate cell adhesion on a typically non-adhesive material, and that a chemical dissimilarity between the topographic features and the background enhances this effect through greater cell-surface interaction. In addition to the aforementioned studies, cell response was also assessed on PNIPAAm thin films coatings with thicknesses ranging from 100 nm to 7 nm. Cell adhesion and spreading was enhanced as the thickness of the thin film decreased. This change was more noticeable below 30 nm, wherein 7 nm shows the highest cell adhesion and spreading enhancement. The results reported are preliminary results and further experiments will be conducted, to support the data. It is believed that cellular response was enhanced due to a change in surface topography at the nanoscale level.

Microsporidia are a distant fungal pathogen that have severe clinical consequences for the immunocompromised. Previous work identified a microsporidian pathogen protein termed Microsporidian ADAM or MADAM. This protein has close sequence homology to other ADAM proteins (A Disintegrin and Metalloproteinase) in two microsporidian species, Encephalitozoon intestinalis and E. cuniculi. ADAM proteins have a wide range of functions, including binding to integrins and host signaling. It is known that many pathogens manipulate integrins to invade host cells, and it is predicted that microsporidia are also exploiting this host target. Previous work with the MADAM protein demonstrated that this protein has a role in adherence to host cells. Separate work showed integrin inhibitors can also decrease spore adherence to cells. Experiments in this project complement previous research and further characterize the binding of microsporidia to host integrins and the intracellular consequences of that binding. This work found the integrin binding sequence of MADAM (MADAM peptide) is important for spore binding to host cells. Separate work shows that the host β1 integrin is also involved in spore adherence. Additional work demonstrated that spores and the MADAM peptide elicited an increase in host integrin signaling in Western blotting experiments. And finally, preliminary acellular interferometry experiments suggest the MADAM protein binds specifically to α5β1 and α6β4 integrins. Together, these results suggest microsporidia spores rely, in part, on host integrins to bind to host cells before infection.

No description available.

The objective of this thesis was to investigate the role of hydroxyapatite and silicate-substituted hydroxyapatite synthetic bone graft substitute (SBG) material properties in modulating the processes of protein adsorption and desorption, and their combined role in the subsequent regulation of cell attachment, proliferation and differentiation on the surfaces of these materials in vitro. As a result of their purported role in promoting osteogenic behaviour in vivo the materials parameters selected for investigation were chemistry (stoichiometric hydroxyapatite (HA) versus 0.8wt% silicate-substituted hydroxyapatite (SA)) and strut porosity (20% versus 30% strut porosity). Cell attachment and response to different SBG was assessed to samples in the ‘as received’ condition as well as after a series of sequentially varied pre-treatments with solutions of phosphate buffered saline or cell culture media either unsupplemented or in combination with mixed serum proteins and/or Fibronectin (Fn). This enabled investigation of the effect of sample chemistry and strut porosity on mixed serum protein interactions and Fn adsorption under both competitive and non-competitive conditions, and the study of subsequent regulation of cell attachment and response as a consequence of pre-treatment. Results showed that serum protein interactions were key to modulation of cell response to chemistry, and there was evidence that for Fn this may be related to conformational changes in the adsorbed protein rather than its level of enrichment in the protein interlayer. In terms of the materials properties investigated strut porosity was found to be the most dominant factor in the regulation of cell response, where SBG with 30% strut porosity promoted human mesenchymal stem cell (hMSC) osteoblastic differentiation. Moreover hMSC response to SBG with 30% strut porosity seemed to be less sensitive to pre-treatment. In conclusion, the results of these experiments indicate that strut porosity more directly influences the cellular response to HA and SA BGS than chemistry in vitro. Moreover, the role that Fn and other serum proteins have in regulating this response is dependent on the physiological environment and BGS chemistry.

The overall objective of this work was to assess the suitability of poly(3-octylthiophene) (P3OT) to sustain MC3T3-E1 osteoblast attachment and growth. The central hypothesis was that specific P3OT film properties (i.e., thickness, film preparation conditions, and level of doping) are able to regulate osteoblast functions (i.e., attachment and proliferation). Discrete and combinatorial techniques were utilized to prepare and characterize thin films of P3OT, a semiconductor in its undoped state, and to study its interaction with MC3T3-E1 osteoblasts. In this work we demonstrate that P3OT is a suitable surface to sustain MC3T3-E1 attachment and proliferation with no observed cytotoxicity. We show that P3OT has an effect on MC3T3-E1 attachment and proliferation as area, circularity, and proliferation ratio are significantly different for P3OT compared to control surfaces. We also demonstrate that P3OT doping and film preparation conditions have an effect on osteoblast attachment and proliferation but that thickness over a low and high range does not affect osteoblast functions. This work is significant because it contributes to the growing area of conducting polymers in biomedical applications and establishes P3OT as a potential cell substrate that sustains MC3T3-E1 attachment and promotes high levels of cell proliferation.

Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were conducted on a limited range of surface textures. Titanium samples were ground to produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.

PMMA is a widely used biomaterial especially in the fields of orthopedia, orthodontia and ophthalmology. When biocompatibility is considered, modification of the biomaterials&amp / #8217 / surface may be needed to optimize interactions of the biomaterial with the biological environment. After the surface modifications one of the most important changes that occur is the change in the surface free energy (SFE). SFE is an important but an obscure property of the material and evaluation methods with different assumptions exist in the literature. In this study, SFE of pristine and oxygen plasma modified PMMA films were calculated by means of numerous theoretical approaches (Zisman, Saito, Fowkes, Berthelot, Geometric and Harmonic Mean and Acid-Base) using numerous liquids and the results were compared to each other to elucidate the differences of methods. Dispersive, polar, acidic and basic components of the SFE were calculated by the use of different liquid couples and triplets with the application of Geometric and Harmonic mean methods and Acid-Base approach. The effect of SFE and the components of SFE on the cell attachment efficiencies were examined by using fibroblast cells. It was observed that with the treatment of oxygen plasma, cell attachment capability and hydrophilicity of PMMA surfaces were altered depending on the applied power and duration of the plasma.