Biologically inspired materials are being developed with the aim of improving the integration of medical implants and minimizing non-desirable host reactions. A promising strategy is the design of topographically patterned surfaces that resemble those found in the extracellular environment. Nanoporous alumina has been recognized as a potential biomaterial and as an important template for the fabrication of nanostructures. In this thesis in vitro studies were done to elucidate the role of alumina nanoporosity on the inflammatory response. Specifically, by comparing alumina membranes with two pore sizes (20 and 200 nm in diameter). Complement and platelet activation were evaluated as well as monocyte/macrophage behaviour. Whole blood was incubated with the alumina membranes and thereafter the biomaterial surfaces were evaluated in terms of protein and platelet adhesion as well as procoagulant properties. The fluid phase was analyzed for complement activation products and platelet activation markers. Besides, human mononuclear cells were cultured on the alumina membranes and cell adhesion, viability, morphology and release of pro-inflammatory cytokines were evaluated. The results indicated that nanoporous alumina with 200 nm pores promotes higher complement activation than alumina with 20 nm pores. In addition, platelet response to nanoporous alumina was found to be highly dependent on the material porosity, as reflected by differences in adhesion, PMP generation and procoagulant characteristics. A clear difference in monocyte/macrophage adhesion and activation was found between the two pore size alumina membranes. Few but highly activated cells adhered to the 200 nm membrane in contrast to many but less activated monocytes/macrophages on the 20 nm surface. The outcome of this work emphasizes that nanotopography plays an important role in the host response to biomaterials. Better understanding of molecular interactions on nano-level will undoubtedly play a significant role in biomaterial implant development and will contribute to design strategies for controlling specific biological events.

<p>Many implanted devices fail due to the formation of an avascular capsule. Fat is known to promote healing and vascularization. It is possible that isolating and attaching ASCs (adipose stem cells) to an implanted device improves the healing in the adjacent tissue. </p><p> Various attachment methods were studied, and the fibronectin treatment was found comparable to or better than other treatments. Next, bare and ASC coated polyurethane were implanted into rats. The fibrous capsule surrounding the bare polyurethane was thicker and contained more collagen at 8 weeks. Additionally, the microvessel density in the tissue surrounding the ASC coated polyurethane was significantly higher at 4 and 8 weeks. Quantification of glucose sensor response following ASC attachment for 1 week found no measurable significant differences in function.</p><p> The bioluminescence technique, which quantifies the tissue glucose concentration around the implant at the moment of freezing, was used to determine if ASC attachment to biomaterials impacts the tissue glucose concentration profile. ASC attachment to polyurethane and to glucose sensors did not significantly change the glucose profiles in the tissue. However, a quantifiable glucose concentration profile was observed around all glucose sensors.</p><p> The final experiments were performed to identify a possible mechanism that adipose tissue uses to alter the foreign body response. In vitro experiments showed that VEGF (VEGF-A specifically) secretion following ASC attachment to polyurethane was 10-20 times higher than with fibroblast attachment after three days and 40-70 times higher after six days. This high secretion of VEGF would likely have in vivo physiological affects on microvasculature.</p><p> In conclusion, the attachment of ASCs to polyurethane reduced the thickness and collagen content of the fibrous capsule surrounding ASC coated implants and increased the microvessel density in adjacent tissue. In addition, ASC attachment did not enhance glucose sensor function, nor did it decrease the glucose concentration in the adjacent tissue. Finally, ASCs were found to secrete high amounts of pro-vascular cytokines, which likely plays a key role in the observed improvement of the foreign body response.</p> / Dissertation

The microstructure and mechanics of collagen and elastin protein fiber networks dictate the mechanical responses of all soft tissues and related organ systems. In this project, we endeavored to meet or exceed native tissue mechanical properties through mimicry of these extracellular matrix components with synthetic collagen fiber and elastin analogues. Significantly, these studies led to the development of a framework for the design and fabrication of protein-based soft tissue substitutes that reproduced many aspects of native biomechanics. A scalable process was developed for production of synthetic collagen microfibers at a rate of 60 m/hr. Fiber properties and ultrastructure were characterized by uniaxial mechanical testing, differential scanning calorimetry, transmission electron microscopy, and second harmonic generation analysis. In vivo responses to synthetic fibers were evaluated in a murine model. A scalable, semi-automated process was designed for the fabrication of multilamellar membranes comprised of sheets of an elastin analogue reinforced with synthetic collagen fibers. Fibers could be organized in a precisely defined three-dimensional hierarchical pattern. The structure of these fiber composites was analyzed by scanning and transmission electron microscopy, and digital volumetric imaging. The effects of fiber orientation and volume fraction on uniaxial mechanical responses were evaluated. Increased fiber volume fraction and alignment increased Young's modulus, resilience, and yield stress. Highly extensible, elastic tissues display a functionally significant transition from low to high modulus deformation at a transition point strain dictated by the crimped collagen microstructure. This response was replicated by the fabrication of dense arrays of microcrimped synthetic collagen fibers embedded in an elastin analogue. The degree of microcrimping could be varied, and generated a transition point mechanical response. Cyclic tensile deformation did not substantially alter microcrimp morphology. A series of small-diameter vascular grafts consisting of an elastin-like protein reinforced with controlled volume fractions and orientations of synthetic collagen fiber was designed and prototyped. The optimal design satisfied target properties with suture retention strength of 173 ± 4 g-f, burst strength of 1483 ± 143 mm Hg, and compliance of 5.1 ± 0.8 %/100 mm Hg.

<p>Today, the majority of materials used for single-use packaging are petroleum-based synthetic polymers. With increased concern about the environmental protection, efforts have been made to develop alternative biodegradable materials from renewable resources. Starch offers an attractive alternative since it is of low cost and abundant. However, the starch material is brittle without plasticizer and the mechanical properties of starch materials are highly sensitive to moisture.</p><p>In nature, the plant cell walls combine mechanical stiffness, strength and toughness despite a highly hydrated state. This interesting combination of properties is attributed to a network based on cellulose microfibrils. Inspired by this, microfibrillated cellulose (MFC) reinforced starch-based nanocomposites films and foams were prepared. Films with a viscous matrix and MFC contents from 10 to 70wt% were successfully obtained by solvent casting. The films were characterized by DSC, DMA, FE-SEM, XRD, mercury density measurements, and dynamic water vapor sorption (DVS). At 70wt% MFC content a high tensile strength together with high modulus and high work of fracture was observed. This was due to the nanofiber and matrix properties, favourable nanofiber-matrix interaction, a good dispersion of nanofibers and the MFC network.</p><p>Novel nanocomposite foams were obtained by freeze-drying aquagels prepared from 8wt% solutions of amylopectin starch and MFC. The MFC content was varied from 10 to 70wt%. For composite foam with MFC contents up to 40wt%, improved mechanical properties were observed in compression. The mechanical properties depended both on the cell wall properties and the cell-structure of the foam. The effect of moisture (20-80% RH) on the dynamical properties of composite foam with 40wt% MFC was also investigated and compared to those of neat starch foam. Improved storage modulus was noted with MFC content, which was a result of the nanofiber network in the cell-wall. In addition, the moisture content decreased with MFC content, due to the less hydrophilic nature of MFC.</p>

Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed.In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair.In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

Cell adhesion to extracellular matrix (ECM) is critical to various cellular processes like cell spreading, migration, growth and apoptosis. At the tissue level, cell adhesion is important in the pathological and physiological processes that regulate the tissue morphogenesis. Cell adhesion to the ECM is primarily mediated by the integrin family of receptors. The receptors that are recruited to the surface are reinforced by structural and signaling proteins at the adhesive sites forming focal adhesions that connect the cytoskeleton to further stabilize the adhesions. The functional roles of these focal adhesions extend beyond stabilizing adhesions and transduce mechanical signals at the cell-ECM interface in various signaling events. The objective of this research is to analyze the role of the spatial distribution of the focal adhesions in stabilizing the cell adhesion to the ECM in relation to cell's internal force balance. The central hypothesis was that peripheral focal adhesions stabilize cell adhesion to ECM by providing for maximum mechanical advantage for resisting detachment as explained by the membrane peeling mechanism. Micropatterning techniques combined with robust hydrodynamic shear assay were employed to test our hypothesis. However, technical difficulties in microcontact printing stamps with small and sparse features made it challenging to analyze the role of peripheral focal adhesions in stabilizing cell adhesion. To overcome this limitation, the roof collapse phenomenon in stamps with small and sparse features (low fill factor stamps) that was detrimental to the reproduction of the adhesive geometries required to test the hypothesis was analyzed. This analysis lead to the valuable insight that the non-uniform pressure distribution during initial contact caused by parallelism error during manual microcontact printing prevented accurate replication of features on the substrate. To this end, the template of the stamp was modified so that it included an annular column around the pattern zone that acted as a collapse barrier and prevented roof collapse propagation into the pattern zone. Employing this modified stamp, the required geometries for the cell adhesion analysis were successfully reproduced on the substrates with high throughput. Adhesive areas were engineered with circular and annular patterns to discern the contribution of peripheral focal adhesions towards cell adhesion strength. The patterns were engineered such that two distinct geometries with either constant adhesive area or constant spreading area were obtained. The significance of annular patterns is that for the same total adhesive area as the circular pattern, the annular pattern provided for greater cell spreading thereby increasing the distance of the focal adhesions from the cell's center. The adhesion strength analysis was accomplished by utilizing hydrodynamic shear flow in a spinning disk device that was previously developed. The results indicate that for a constant total adhesive area, the annular patterns provide for greater adhesion strength by enhancing cell spreading area and providing for greater moment arm in resisting detachment due to shear. The next examination was the effect of the cell's internal force balance in stabilizing the cell adhesion. The working hypothesis was that microtubules provide the necessary forces to resist the tensile forces expressed by the cell contractile machinery, thereby stabilizing cell adhesion. Since microtubule disruption is known to enhance cell contractility, its effect on the cell adhesion strength was examined. Moreover, the force balance in cells was altered by engineering adhesive areas so that the cells were either spherical or completely spread and then disrupted microtubules to understand the significance of the force balance in modulating the cell adhesion strength. The results indicated that disruption of microtubules in cells on adhesive islands resulted in a 10 fold decrease in adhesion strength compared to untreated controls whereas no significant change was observed in completely spread cells between treated and untreated controls. This is in surprising contrast to the previous contractility inhibition studies which indicate a less pronounced regulation of adhesion strength for both micropatterned and spread cells. Taken together, these findings suggest that the internal force balance regulated by cell shape strongly modulates the adhesion strength though the microtubule network. In summary, this project elucidates the role of peripheral focal adhesions in regulating the cell adhesion strength. Furthermore, this study also establishes the importance of the internal force balance towards stabilizing the cell adhesion to the ECM through the microtubule network.

The mechanisms of interaction between inorganic matter and biomolecules, as well as properties of resulting hybrids, are receiving growing interest due to the rapidly developing field of bionanotechnology. The majority of potential applications for metal-biohybrid structures require stability of these systems under vacuum conditions, where their chemistry is elusive, and may differ dramatically from the interaction between biomolecules and metal ions in vivo. Here we report for the first time a photoemission and X-ray absorption study of the formation of a hybrid metal-protein system, tracing step-by-step the chemical interactions between the protein and metals (Cu and Fe) in vacuo. Our experiments reveal stabilization of the enol form of peptide bonds as the result of protein-metal interactions for both metals. The resulting complex with copper appears to be rather stable. In contrast, the system with iron decomposes to form inorganic species like oxide, carbide, nitride, and cyanide.

High linoleic acid soybeans facilitate maximum production of soy-based polyurethane. The objectives of this study were to: 1) Evaluate environmental influence on yield and seed composition traits; 2) Estimate correlation coefficients between linoleic acid with agronomic traits; 3) Validate SSR markers associated with fatty acid QTL in multiple environments and across diverse genotypes; and 4) Evaluate the influence of fertilizers differing in P and K concentrations on seed fatty acids. RG25 was identified as the best genotype to be commercialized for polyurethane production. Strong marker-trait associations across environments included Satt_335, Satt389, Satt556 associated with palmitic and stearic, Satt389 with oleic, Satt389 and Satt537 with linoleic acid. A significant increase in linoleic acid content was observed when plants received modified Hoagland’s solution with 2×K compared to without K. Development of a high linoleic acid soybean line for polyurethane production is feasible using validated SSR markers and high K fertility. / Ontario Ministry of Agriculture, Food and Rural Affairs

Titanium and titanium based alloys are widely used in dentistry and orthopedics to replace hard tissue and to mend broken bones. It has become a material of choice due to its low density, high strength, good biocompatibility and its capacity to integrate closely with the bone. Today, modern materials and surgical techniques can enable patients to live longer, and aid in maintaining or regaining mobility for a more fulfilling life. There are, however, instances where implants fail, and one of the primary causes for implant failure is infection. This thesis deals with two possible ways of reducing or eliminating implant associated infections; TiO2 photocatalysis, where a surface can become antibacterial upon irradiation with UV light; and incorporation of silver, where a subsequent release of silver metal ions result in an antibacterial effect. For the TiO2 photocatalysis strategy, a simple and cost effective chemical oxidation technique, using hydrogen peroxide (H2O2) and water, was used to create an active TiO2 surface on titanium substrates. This surface was shown to effectively degrade an organic model substance (rhodamine B) by generating reactive oxygen species (ROS) under UV illumination. However, it was shown that Ti-peroxy radical species remaining in the surface after the H2O2-oxidation process, rather than generation of ROS from a heterogeneous photocatalytic process, was responsible for the effect. This discovery was further exploited in a TiO2/H2O2/UV system, which demonstrated synergy effects in both rhodamine B degradation tests and in antibacterial assays. For the silver ion release strategy, a combinatorial materials science approach was employed. Binary Ag-Ti oxide gradients were co-deposited in a reactive (O2) environment using a custom built physical vapor deposition system, and evaluated for antibacterial properties. The approach enabled synthesis and composition-structure-property evaluation unlikely to have been achieved by traditional means, and the gradient coatings demonstrated antibacterial properties against both S. aureus and S. epidermidis according to silver ion release. The release was shown to depend more on structural features, such as surface area, crystallinity and oxidation state, than on composition. Ag-Ti oxide gradients were also evaluated under UV illumination, as Ag deposits on crystalline TiO2 can enhance photocatalytic properties. In this work, however, the TiO2 was amorphous and UV illumination caused a slight reduction in the antibacterial effect of silver ions. This was attributed to a UV-induced SOS response in the S. epidermidis bacteria. The results of this thesis demonstrate that both TiO2 photocatalysis, or UV induced activation of Ti-peroxy radical species, as well as incorporation of silver are viable antibacterial strategies for titanium biomaterials. However, their clinical applications are still pending risk-benefit analyses of potential adverse host tissue responses.

<p>Extending the functional lifetime of acrylic poly(methyl methacrylate) (PMMA) bone cement may reduce the number of revision total joint replacement (TJR) surgeries performed each year. We developed a system utilizing an encapsulated water-reactive, FDA-approved tissue adhesive, 2-octyl cyanoacrylate (OCA), as a healing agent to repair microcracks within a bone cement matrix. The proposed research tested the following hypotheses: (1) reactive OCA can be successfully encapsulated and the resulting capsules thoroughly characterized; (2) the static mechanical properties of the PMMA composite can be improved or maintained through inclusion of an optimal wt% of OCA-containing capsules; (3) PMMA containing encapsulated OCA has a prolonged lifetime when compared with a capsule-free PMMA control as measured by the number of cycles to failure; and (4) the addition of capsules to the PMMA does not significantly alter the biocompatibility of the material. Based on the experiments reported herein, the primary conclusions of this dissertation are as follows: (1) functional OCA can be encapsulated within polyurethane spheres and successfully incorporated into PMMA bone cement; (2) lower wt% of capsules maintained the tensile, compressive, fracture toughness, and bending properties of the PMMA; (3) inclusion of 5 wt% of OCA-containing capsules in the matrix increased the number of cycles to failure when compared to unfilled specimens and those filled with OCA-free capsules; and (4) MG63 human osteosarcoma cell proliferation and viability were unchanged following exposure to OCA-containing PMMA when compared with a capsule-free control.</p> / Dissertation