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All artificial organ systems and medical devices that operate in direct contact with blood elicit activation of coagulation and platelets, and their long-term use often necessitates antithrombotic therapies that carry significant cost and bleeding risk. Thrombomodulin (TM) is a major endogenous inhibitor of blood coagulation localized on the endothelial cell surface. The overall objective of this research is to develop clinically durable synthetic materials by incorporating TM as a solid-supported film to actively and sustainably attenuate thrombus formation at the blood-contacting interface. During the course of this research, we developed site-specific approaches to covalently attach TM on the luminal surface of commercial vascular grafts using bioorthogonal chemistry that was compatible with ethylene oxide sterilization. Notably, we demonstrated the superior efficacy of TM to reduce platelet deposition compared with commercial heparin modified grafts using a non-human primate model of acute graft thrombosis. Finally, we optimized a novel reversible chemistry to rapidly and repeatedly regenerate immobilized TM, with the potential to significantly extend the lifetime of biologically active films.

Bone consists of collagen/hydroxyapatite (HA) composites in which poorly crystalline carbonated calcium phosphate is intercalated within the fibrillar structure. Normal bone mineral is a carbonated-apatite, but there are limited data on the effect of mineral containing carbonate on cell response. Although the exact biological role of silicate in bone formation is unclear, silicate has been identified at trace levels in immature bone and is believed to play a metabolic role in new bone formation. To mimic the inorganic and organic composition of bone we have developed a variety of bone graft substitutes. In the present body of research, we characterized the surface composition of human cortical and trabecular bone. When then characterized the surface compositions of the following potential bone substitutes: carbonated hydroxyapatite (CO₃²-HA), silicated hydroxyapatite (Si-HA), and collagen sponges mineralized with calcium phosphate using the polymer-induced liquid-precursor (PILP) process. In the latter substitutes, the PILP process leads to type I collagen fibrils infiltrated with an amorphous mineral precursor upon which crystallization leads to intrafibrillar HA closely mimicking physiological bone mineral. We then determined the osteoblast-like cell response to each bone substitute to characterize the substrate’s effect on osteoblast differentiation. The observations collectively indicate that cells are sensitive to the formatting of the mineral phase of a bone substitute and that this format can be altered to modulate cell behavior.

In nature, organisms have long been able to create elaborate mineral structures at ambient temperatures. From a materials science and engineering perspective, favorable properties emerge when the synthesis process can be controlled at finer levels. New strategies in materials chemistry synthesis has been inspired by biomineralization: biomimetics. In this work, silk fibroin films were used to synthesize gold nanoparticles room temperature by soaking a free standing 15nm silk film in HAuCl4. Particles ranged in size and shape from 5nm spheres to 105nm hexagons. Secondly, a film of ZnO1 peptide (ZnO selectively binding peptide) was successfully formed by drop casting on both silk and polystyrene surfaces. Using a HMT + Zn(NO3)2 system for ZnO wet chemical deposition, rods were formed on the peptide surface. Changing solution concentration and growth time affected the density and size of the nanorods. Spin coating a 3nm peptide film reduced the roughness to <1nm, upon which an array of vertical ZnO rods with controllable density was synthesized.

Biomaterial surface properties such as microtopography and energy can change cellular responses at the cell-implant interface. Phospholipase D (PLD) is required for differentiation of osteoblast-like MG63 cells on machined and grit-blasted titanium surfaces. Here, we determined if PLD is also required on microstructured/high-energy substrates and the mechanism involved. shRNAs for human PLD1 and PLD2 were used to silence MG63 cells. Wild-type and PLD1 or PLD1/2 silenced cells were cultured on smooth-pretreatment surfaces (PT); grit-blasted, acid-etched surfaces (SLA); and SLA surfaces modified to have higher surface energy (modSLA). PLD was inhibited with ethanol or activated with 24,25-dihydroxyvitamin-D₃ [24R,25(OH)₂D₃]. As surface roughness/energy increased, PLD mRNA and activity increased, cell number decreased, osteocalcin and osteoprotegerin increased, and protein kinase C (PKC) and alkaline phosphatase specific activities increased. Ethanol inhibited PLD and reduced surface effects on these parameters. There was no effect on these parameters after knockdown of PLD1, but PLD1/2 double knockdown had effects comparable to PLD inhibition. 24R,25(OH)₂D₃increased PLD activity and production of osteocalcin and osteoprotegerin, but decreased cell number on the rough/high-energy surfaces. These results confirm that surface roughness/energy-induced PLD activity is required for osteoblast differentiation and that PLD2 is the main isoform involved in this pathway. Here we showed that PLD is activated by 24R,25(OH)₂D₃ in a surface-dependent manner and inhibition of PLD reduced the effects of surface microstructure/energy on PKC, suggesting that PLD mediates the stimulatory effect of microstructured/high-energy surfaces via PKC-dependent signaling.

This thesis targets cyclic peptide supramolecular structures for biomaterial applications. The introduction gives a brief insight into supramolecular interactions, peptides, and their application in biomaterials. These supramolecular interactions range from the weak forces of electrostatics and van der Waals interactions, to hydrogen bonding and metal-coordination. The application of peptides and supramolecular interactions has become a highly studied area of chemistry, which has quickly gotten attention in the area of biomaterials. The use of peptides in biomaterials seems obvious since in vivo rejection of this material might be limited. Nature can be used as a blue print to direct the path for hydrogen bonding motifs and metal-coordinating interactions and can be applied potentially towards supramolecular biomaterials. Finally, the introduction reviews the use of cyclic peptides and accounts for the synthetic design of the cyclic octapeptide to be used throughout the thesis work. The second chapter of the thesis provides the details by which the synthetic scheme for creating the linear peptides of interest and ultimately the cyclic peptides is described in detail. Many synthetic challenges were met and overcome during this thesis work; the most notable was overcoming purification challenges and poor amino acid coupling reactions that resulted in low yields. This thesis focuses primarily on the di-substituted pyridylalanine cyclic octapeptide, however much of the initial work on the mono-substituted cyclic octapepide was carried out in tandem allowing for comparison of the two peptides necessary for future work.

Implanted combination devices comprising both biological as well as biomaterial components may trigger non-specific inflammatory responses against the biomaterial component as well as specific immune responses against the biological component. This specific immune response may be enhanced by the biomaterial, thereby implying a biomaterial-mediated adjuvant effect, or in contrast may be mitigated by the biomaterial. Since adjuvants function by triggering dendritic cell (DC) maturation, biomaterials may regulate DC responses and hence facilitate DC-orchestrated host responses. This research work has focused on examining DC responses to different model self-assembled monolayer (SAM) biomaterial chemistries, as an in vitro readout of the potential of these biomaterials to trigger DC maturation. The underlying hypothesis was that DCs recognize and respond to biomaterials either indirectly through the adsorbed protein layer, specifically through carbohydrate modifications of these proteins, or through carbohydrates inherent in the biomaterial chemistry, using PRRs to initiate an immune response. Towards this goal, DCs were derived from human peripheral blood mononuclear cells (PBMCs) by culture with DC differentiation cytokines and the culture systems were characterized as being composed of DCs as well as associated T and B lymphocytes. Culture of DCs on different SAM chemistries implied differential DC responses in terms of morphology, maturation marker expression and allostimulatory capacities as well as distinct underlying mechanisms responsible for these responses. Enzyme-linked lectin (ELLA) assays were used to characterize the profiles of carbohydrates associated with serum/plasma proteins adsorbed to different SAM chemistries. Differential profiles of DC carbohydrate ligands of CLRs were present on different chemistries. Furthermore, the profiles of human serum/plasma proteins adsorbed to and eluted from different SAM chemistries were assessed using immunoblot analysis. Finally, to observe the roles of carbohydrates in supporting DC maturation in the presence of a biomaterial, DCs were cultured in the presence of partially de-glycosylated FBS from which DC carbohydrate ligands were selectively removed. This research is significant towards the ultimate development of optimal design criteria for biomaterials for use in diverse tissue-engineering or vaccine development applications for which a wide spectrum of adjuvant effects are required.

[Truncated abstract] The synthetic hydrogel poly(2-hydroxyethyl methacrylate) (PHEMA) has been used as a biocompatible biomaterial in ocular devices, such as soft contact lenses, intraocular lenses and an artificial cornea. Due to its favourable properties as an already established (but non-biodegradable) biomaterial, PHEMA is an interesting candidate for use as a material for scaffolds in tissue engineering. A tenant of tissue engineering scaffolds is obtaining the appropriate porous morphology to allow for successful cellular attachment and support. PHEMA hydrogels exhibit varied morphological features, which range from non-porous (homogeneous) to macroporous (heterogeneous) and can be readily obtained by fine-tuning the polymerisation conditions. A desirable feature for matrices that are to be used as tissue supports is the ability to biodegrade in a biological environment. This thesis describes the preparation and enzymatic biodegradation behaviour of novel porous PHEMA hydrogels that have been crosslinked with biodegradable peptide-based crosslinking agents. Peptide-based crosslinking agents were designed to contain two terminal polymerisable groups flanking an internal biodegradable backbone. This backbone was specifically designed to be targeted by the proteolytic enzyme papain. The general design template allowed for the development of a synthetic methodology that was readily implemented for the production of a range of olefin-peptide conjugates. A suite of olefin-peptide conjugates of general structure I were synthesised, characterised and further tested with papain to determine their biodegradation properties. ... The second strategy for producing bioresorbable degradation fragments involved the incorporation of the highly hydrophilic comonomer, poly(ethylene glycol) PEG into the PHEMA backbone. The addition of PEG to PHEMA resulted in the formation of homogeneous hydrogels that had an improved hydrophilicity compared to their heterogeneous PHEMA counterparts. The synthetic conditions for the preparation of PHEMA and PHEMA-co-PEG hydrogels by photoinitiated polymerisation were thoroughly investigated. It was found that the pore morphology and general properties (non-porous to macroporous) of these hydrogels could be controlled by the appropriate choice of polymerisation conditions. The hydrogels were characterised by scanning electron microscopy, thermal gravimetric analysis and differential scanning calorimetry. The peptide-based crosslinking agents were successfully co-polymerised with the HEMA and PEGMA via photoinitiated polymerisation to provide a range of PHEMA and PHEMA-co-PEG hydrogels that displayed both homogeneous and heterogeneous hydrogel properties. The final crosslinked hydrogels were characterised by scanning electron microscopy and were subjected to enzymatic hydrolysis. The PHEMA-peptide conjugate hydrogels proved to be biodegradable, with degradation behaviour dependent on the hydrogel formulation and the length of the peptide-based crosslinking agent.

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: John L. Brash. Includes bibliographical references. Also available online.

Thesis (Ph.D.)--Flinders University, School of Chemistry, Physics and Earth Sciences. / Typescript bound. Includes bibliographical references and list of publications. Also available online.