Endothelial colony forming cells (ECFCs) are circulating cells able to differentiate into mature endothelial cells and replenish the endothelial lining at the sites of vascular damage. Their utilization for cell therapies aiming to restore healthy endothelial lining of blood vessels and stimulate neovascularization of ischemic tissues has been the object of intense investigation. The overall aim of this project was to investigate and develop novel approaches for the promotion of vasculogenesis and endothelisation of vascular grafts by ECFCs. First, protease-activated receptors (PARs) were investigated as potential targets to stimulate in ECFC-driven vasculogenesis and promote therapeutic revascularization. Our data showed that PAR-1 and PAR-2 are both expressed in ECFCs and functionally coupled to the ERK1/2 pathway. Specific stimulation of PAR-1, but not PAR-2, significantly inhibits in vitro tube formation by ECFCs, and this effect is due to the down-regulation of VEGFR-2. Although the role of PAR-2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade. Secondly, we investigated the use of human platelet lysate gel (hPLG) as an animal product-free and patient-specific tool to isolate, amplify, differentiate and deliver endothelial progenitor cells. This extracellular matrix (ECM) was able to support the proliferation of ECFCs in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Interestingly, the culture of ECFCs on hPLG led to the upregulation of several angiogenic genes, such as VEGFR-2 and PDGFR-β, and also induced the robust sprouting of existing vessels in an ex vivo model. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. Finally, the biomimetic and pro-angiogenic properties of human platelet lysate (hPL) were utilised to facilitate adhesion and proliferation of ECFCs on polymeric materials. hPL was shown to promote endothelisation of biomaterials, which can be utilised for tissue engineering purposes. Novel electrospun polymeric tubular scaffolds were developed and their surface properties enhanced using plasma treatment. These scaffolds exhibit increased adsorption of proteins from hPL, which acted as an interfacial layer to promote the adhesion and proliferation of ECFCs on their surface. Such findings demonstrate that the pro-angiogenic and pro-vasculogenic properties of platelet-derived factors can be transferred to scaffolds to stimulate endothelial growth on synthetic scaffolds for tissue engineering without the use of recombinant or animal products. In conclusion, we propose the use of ECFCs with platelet-derived products as an ideal synergy for the vascularization of tissue engineered constructs.

There has been much work into getting clear and precise images of bone growing within different osteoconductive and osteoinductive scaffolds for the aim of investigating and quantifying the effect the different grafts have on the bone that forms within the graft. Before the bone structure and volume can be quantified, the images produced need to segmented into their different regions. Using images produced from x-ray computed tomography, the samples can be segmented based on their densities. As the voxels have distinct size, if just the density is used to segment out the regions, there will be some miss-identification at the edges of the regions (ghosting). To overcome this problem of misidentification, automated segmentation methods were developed which take not only the intensity of the voxels in the images (which are related to the density) into account for the segmentation but also the local properties. With correct segmentation the volume and surface area are better represented and methods for structure measurement can and were developed. These methods allow for not only the structure of the bone and implants to be quantified, but for the change in structures between the different implants to be compared. This allows for the different structures caused by the different graft materials to be seen and compared. This comparison when used on its own or with other methods such as histology not only allows for the different structures to be identified but all the change in structures due to factors such as remodelling to be identified.

Thiol-ene radical coupling is increasingly used for the biofunctionalisation of biomaterials and the formation of 3D hydrogels enabling cell encapsulation. Indeed, thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions of peptides or proteins, in particular in situ, during cell culture or encapsulation: thiol-ene coupling occurs specifically between a thiol (from cysteine residues for example) and a non-activated alkene (unlike Michael addition); it is relatively tolerant to the presence of oxygen; it can be triggered by light, to trigger dynamic systems or for patterning. Despite such interest, little is known about the factors impacting thiol-ene chemistry in situ, under biologically relevant conditions. Here we explore some of the molecular parameters controlling photo-initiated thiol-ene coupling chemistry with a series of alkenes and thiols, including peptides, in buffered conditions. 1H NMR spectroscopy and HPLC were used to quantify the efficiency of couplings and the impact of the intensity of UV exposure, pH of the buffer, as well as the molecular structure and local microenvironment close to alkenes and thiols to be coupled. Our studies demonstrate that molecular design should be carefully selected in order to achieve high biofunctionalisation levels in biomaterials with peptides.

Poly(lactic acid) / Cellulose Nanocrystals (PLA / CNs) were simultaneously electrospun to fabricate a novel renewable and biocompatible nanocomposite as potential scaffold for bone tissue engineering. CNs were successfully incorporated into the PLA fibers to reinforce the electrospun fiber mat. Thermal, chemical and mechanical analyses were performed to characterize and determine the properties of the scaffold fabricated. Highly porous fibers with fibers diameters in the range of 500-1000 nm were characterized by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Crystallinity of the electrospun nanocomposite was studied by Differential Scanning Calorimetry. Adipose-derived human mesenchymal stem cells (hMSCs) were used to study the cytocompatibility of the nanocomposite scaffold. Life/dead cell assay was performed to determine cell viability of the scaffolds. After one week of cell culture, confocal microscopy indicated that the cells grown on the PLA / CNs nanocomposite were confluent and very well aligned along the fibers while cells cultured on pure PLA fibers were not as confluent as in the developed nanocomposite. This project has demonstrated the feasibility of the fabricated PLA/CNs nanocomposite as a potential scaffold for bone tissue engineering.

Hydrogels are cross-linked networks of highly hydrophilic polymer chains. When reduced to colloidal dimensions, particles of this sort are termed “microgels�? and both discrete particles and ensembles have intriguing properties. Microgels can be made to be susceptible to numerous environmental stimuli, such as temperature and pH. The resultant changes in the network hydration lead to characteristic swelling responses which can have great impact on properties of the gel network such as the porosity, hydrophilicity, stiffness, or particle-particle packing. The multitude of responsive stimuli; the architectural versatility of discrete particles; and the variety of particle ensembles have made the study of microgels and their assemblies a very rich field. Primarily due to their physiological softness and the aforementioned versatility, responsive microgels are of great interest as a material to address the daunting challenges facing the next generation of healthcare. This dissertation describes investigations into hydrogel nanoparticles and assemblies thereof, with the goals of expanding their utility in applications such as drug delivery and non-fouling interfaces through the development of novel materials to both extend their synthetic versatility and to probe their underlying properties. Physiologically-relevant degradable cross-linking within microgels is studied, though the incorporation of hydrolytically degradable or reduction-responsive cross-links. More complex structures are demonstrated for both types of cross-linking as synthetic and architectural control enables additional functional microgel designs. Microgel assemblies, particularly thin films, have been demonstrated to have numerous desirable properties for biological applications, such as reduced cell attachment, drug delivery, and self-healing capabilities. This dissertation includes additional fundamental studies of microgel films, both in their synthesis, such as methods for depositing films onto colloidal substrates, and in their application, as investigations into the origins and critical factors for self-healing films. Further, the cell-resistant properties of microgel multilayers are studied and evidence suggests that the viscoelastic or mobile character of the films is likely the main factor that directs cell adhesion. The work in this dissertation serves to both expand our toolset with regard to the functional synthesis of microgels and assemblies and to improve our fundamental understanding of phenomena of interest for a variety of potential applications. Both of these should serve to allow the enormous potential of hydrogel nanoparticles and their assemblies to be more efficiently tapped for a wide range of applications in the field of biomaterials.

Cells rely on time-dependent binding and activation by the ECM to initiate downstream signal transduction. It is unknown whether adhesion to a ligand is required throughout various cell processes, or only during a specified time period ("temporal threshold”). Current approaches to ligand presentation often comprise of static, constant densities of ligands. In contrast, natural cell adhesive interactions with ECMs exhibit spatiotemporal patterns of binding and activation. Therefore, a key to future research in controlling cell-material interactions will be the development of materials that can respond to external stimuli. The objective of this project is to engineer biomaterials that present a UV-labile caged-Arginine-Glycine-Aspartic Acid (RGD) ligand and evaluate the effects on cell activities. RGD is the minimal adhesive sequence of fibronectin. By dynamically modulating adhesive ligand presentation, the effects of temporal control on cell processes can be elucidated. In this caged-peptide, a photo-labile group adjacent to the aspartic acid residue of RGD effectively “masks” a cyclo(RGDfk) peptide. Upon UV irradiation (360 nm), the caging group is released thereby restoring the adhesive activity of the peptide. By having unparalleled spatiotemporal control of RGD ligand presentation, we demonstrated two novel tools for discovery: 1) in vivo ligand presentation to probe downstream tissue behavior and cell infiltration to biomaterial implants, and 2) in vitro ligand presentation in situ using confocal-based live cell microscopy to investigate real-time vinculin recruitment and cell traction force generation. These studies represented the first demonstration of triggerable adhesive ligand presentation in vivo and demonstrated the utility of caged-compounds for probing specific receptor-ligand responses on highly defined PEG-based hydrogels. Triggerable in vitro ligand presentation, combined with traction force microscopy, demonstrated a new research tool for investigating focal adhesion formation and downstream force generation. Taken in whole, these results provide previously unknown insights into the importance of spatiotemporal control of adhesive ligands and created novel new research platforms for future discovery.

When routine repair mechanisms fail to regenerate severe burn wounds, mesenchymal stem cell therapy is considered. However, engrafted mesenchymal stem cells are prone to become myofibroblasts when exposed to high mechanical tension and pro-fibrotic cytokines in the wound microenvironment. Myofibroblast activity increases wound stiffness and activates healthy precursor cells into destructive phenotype, resulting in pathological remodelling and hypertrophic scarring. Using soft silicone substrates with near-physiological stiffness, I tested the hypothesis that myofibroblast characteristics acquired by mesenchymal stem cells in cell culture are preserved by microRNA modifications typical for fibrosis and demonstrated that priming mesenchymal stem cells on soft substrates protect them from subsequent activation and that the mechanically propagated myofibroblast memory is mediated by miR-21. This study aims to demonstrate that suppressing myofibroblast activation will maximize and prolong the beneficial regenerative effects of mesenchymal stem cells while terminating harmful and excessive tissue remodelling characteristic for fibrosis upon engraftment.

When routine repair mechanisms fail to regenerate severe burn wounds, mesenchymal stem cell therapy is considered. However, engrafted mesenchymal stem cells are prone to become myofibroblasts when exposed to high mechanical tension and pro-fibrotic cytokines in the wound microenvironment. Myofibroblast activity increases wound stiffness and activates healthy precursor cells into destructive phenotype, resulting in pathological remodelling and hypertrophic scarring. Using soft silicone substrates with near-physiological stiffness, I tested the hypothesis that myofibroblast characteristics acquired by mesenchymal stem cells in cell culture are preserved by microRNA modifications typical for fibrosis and demonstrated that priming mesenchymal stem cells on soft substrates protect them from subsequent activation and that the mechanically propagated myofibroblast memory is mediated by miR-21. This study aims to demonstrate that suppressing myofibroblast activation will maximize and prolong the beneficial regenerative effects of mesenchymal stem cells while terminating harmful and excessive tissue remodelling characteristic for fibrosis upon engraftment.

The freshwater zebra mussel Dreissena polymorpha is a biofouling species that adheres to varied substrates underwater using a proteinaceous byssus that consists of a bundle of threads tipped with adhesive plaques. This underwater adhesion is an inspiration for the development of medical and dental bioadhesives, however, the byssus is highly resistant to biochemical characterization owing to extensive cross-linking and therefore, limited information is available on the mechanisms of adhesion and cohesion of byssal proteins. We report here on the identification and sequence analysis of eight novel byssal proteins identified in the soluble extract and insoluble matrix from induced, freshly secreted byssal threads with minimal cross-linking, using gel electrophoresis and LC-MS/MS sequencing techniques. Identified byssal proteins have theoretical molecular weights ranging from 4.1 kDa to 20.1 kDa and isoelectric points ranging from 4.2 to 9.6 and have several common characteristics including consensus repeat patterns, block structures and defined sequence motifs.

The freshwater zebra mussel Dreissena polymorpha is a biofouling species that adheres to varied substrates underwater using a proteinaceous byssus that consists of a bundle of threads tipped with adhesive plaques. This underwater adhesion is an inspiration for the development of medical and dental bioadhesives, however, the byssus is highly resistant to biochemical characterization owing to extensive cross-linking and therefore, limited information is available on the mechanisms of adhesion and cohesion of byssal proteins. We report here on the identification and sequence analysis of eight novel byssal proteins identified in the soluble extract and insoluble matrix from induced, freshly secreted byssal threads with minimal cross-linking, using gel electrophoresis and LC-MS/MS sequencing techniques. Identified byssal proteins have theoretical molecular weights ranging from 4.1 kDa to 20.1 kDa and isoelectric points ranging from 4.2 to 9.6 and have several common characteristics including consensus repeat patterns, block structures and defined sequence motifs.