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Chronic wounds have a detrimental impact on patient quality of life, a significant economic cost, and often lead to severe outcomes such as amputation, sepsis or death. The elaborate interplay between bacteria, cutaneous cells, immune cells, growth factors, and proteases in chronic wounds has complicated the development of new therapies that could improve outcomes for chronic wound patients. Existing in vitro models of chronic wounds do not appreciably mimic the complexity of the wound environment. In this work, tissue-engineered skin was developed with the goal of creating an in vitro platform appropriate for testing potential clinical therapies for chronic wounds. The Lumen-X, a digital light processing bioprinter, was used to create tissue-engineered skin from a 7.5% (w/v) gelatin methacryloyl hydrogel laden with primary dermal fibroblasts. This dermal layer was developed with an emphasis on providing a favourable microenvironment for the fibroblasts in order to mimic their in vivo phenotype. An epidermal layer of human keratinocytes was formed on the hydrogel surface and stratified through culture at the air-liquid-interface. The maturation of the epidermis was thoroughly characterized with histology, immunohistochemistry, and trans-epithelial electrical resistance analyses which showed a degree of maturation suitable for wound healing studies. To verify the suitability of this tissue-engineered skin for studying healing in vitro, sharp tweezers were used to create physical wounds in the epidermis which were then infected with Pseudomonas aeruginosa. Reepithelialisation, the production of the pro- inflammatory cytokine TNF-α, and the presence of bacteria were monitored over time, showing healing in wounds without infection and those treated with antibiotics, and potential biofilm formation in infected wounds. The tissue-engineered skin developed here is suitable for use as an in vitro model of the infected chronic wound environment. Future work includes developing better methods for creating the physical wound and characterizing the bacterial biofilm in order to improve the reproducibility and clarity of results. Such a model will then be well-poised to begin testing potential chronic wound therapies in vitro. / Graduate / 2023-07-26

Articular cartilage is a highly specialized connective tissue in the body responsible for protecting and cushioning bony ends in diarthrodial joints. Despite the unique ability of this tough, spongy matrix to absorb repetitive stress and loading, cartilage damage is a common occurrence, and as cartilage possesses poor self-repair capabilities, tissue-engineered cartilage replacement is under development as a viable method of repair. Tissue-engineered constructs have thus far been unable to replicate the matrix composition of native cartilage satisfactorily enough to produce usable mechanical properties; in particular, collagen content is very low. One means of improving engineered construct composition may be pulsed low-intensity ultrasound (PLIUS), which is used clinically to stimulate healing of chronic bone lesions, and has been shown to affect chondrocytes in cartilage explants and engineered constructs. We believe it may be of use specifically in improving collagen quantity and quality in engineered constructs. FT-IR spectroscopy shows promise as a valuable tool in collagen crosslink maturity analysis, replacing the current expensive, complicated standard of HPLC and allowing for high-resolution spatial mapping of components. A spectral parameter has been established in literature as being related to collagen maturity in bone, which we explore as a potential means of assessing collagen quality in our engineered cartilage. The specific aims of this research are twofold: first, to assess whether PLIUS improves primary bovine chondrocyte-seeded poly-glycolic acid (PGA) mesh scaffold composition by culturing groups with and without PLIUS stimulation, and second, to correlate FT-IR parameters (including the aforementioned maturity parameter) from engineered cartilage specimens and pure crosslink peptides to mechanical testing in unconfined compression. / Bioengineering

Articular cartilage has limited intrinsic healing capacity due to its dense and avascular structure. Clinical approaches have been developed to address the limitations associated with the poor ability of articular cartilage to regenerate. Current clinically approved techniques, however, can result in repair tissue that lacks appropriate hyaline cartilage biochemical and biomechanical properties, which lead to uncertain long-term clinical outcomes. Using tissue engineering strategies and a range of scaffolding materials, cell types, growth factors, culture conditions, and culture times, engineered tissues have been produced with compositional and biomechanical properties that approximate that of native tissue. In these studies, a considerable number of samples are typically sacrificed to evaluate compositional and mechanical properties, such as the amount of deposited collagen and sulfated glycosaminoglycan (sGAG) in the constructs. The number of sacrificed samples, as well as the amount of time and resources spent to evaluate the sacrificed samples using current gold standards, motivates an alternative method for evaluation of compositional properties. Vibrational spectroscopy, including infrared, has been considered as an alternative technique for assessment of tissues over the last 15-20 years. Infrared spectroscopy is based on absorbance of infrared light by tissue functional groups at specific vibrational frequencies, and thus, no external contrast is required. Vibrational spectroscopy is typically performed in two frequency regions, the mid infrared region (750-4000 cm-1), where penetration depth is limited to approximately 10 microns, and the near infrared (NIR) region (4000-12000 cm-1). In the NIR region, penetration of light is on the order of millimeters or centimeters, which makes it ideal for obtaining data through the full depth of engineered constructs. Here we employ NIR spectroscopy to nondestructively monitor the development of tissue-engineered constructs over culture period. / Bioengineering

This thesis investigates new processing methods and bone tissue engineering applications of cross-linked cellulose nanocrystal (CNC) aerogels. Aerogels are highly porous, low-density materials that have been praised for their high surface area and interconnected pores, but criticized for their brittleness. This prompted a search for new aerogel “building blocks” to produce more flexible materials; CNCs meet this need and chemically cross-linked CNC aerogels have good compressive strength and shape recovery properties in air and liquid environments. CNCs are high aspect ratio, non-toxic and renewably-sourced nanoparticles. Literature has demonstrated CNC aerogel production using cryo-templating with controlled drying. In this work, we produce aerogels using a new scalable process called pressurized gas expansion (PGX) and compare them to conventional cryo-templated aerogels. PGX aerogels were found to have more expanded fibrillar morphology, a range of mesopore sizes and smaller macropores, in contrast to cryo-templated aerogels that had a sheet-like morphology surrounding larger macropores. Additionally, PGX aerogels had higher specific surface area and porosity, but lower compressive strength due to a lower cross-link density. While neither CNC aerogel type dispersed in water, PGX aerogels partially shrank whereas cryo-templated aerogels did not; this is attributed to their morphological differences. This work shows that new aerogel processing methods can introduce new properties and thus broaden the potential applications of CNC aerogels. One specific biomedical application was evaluated for CNC aerogels – their use as bone tissue scaffolds. Cryo-templated aerogels comprised of CNCs with different surface chemistries, either sulfate or phosphate groups, were found to have attractive chemical, physical and mechanical properties for bone tissue engineering. This work shows that both types of CNC aerogels can facilitate cell proliferation, favorable differentiation, and can nucleate uniform hydroxyapatite growth. These positive in vitro results and the bimodal pore morphology of CNC aerogels make them promising bone scaffolds for in vivo studies. / Thesis / Master of Applied Science (MASc) / Aerogels are light, porous, sponge-like materials that are essentially 99% air by volume. In this work, the aerogels are made from non-toxic plant-based nanoparticles called cellulose nanocrystals (CNCs). This thesis investigates: 1) new ways to control CNC aerogel properties and pore size through different processing methods and 2) the use of CNC aerogels to aid in the repair of damaged bones. High-resolution microscopy and nano-characterization tools show that CNC aerogels have tunable properties, which may extend their possible applications. The internal structure, sponge-like mechanical properties and biocompatibility of CNC aerogels allowed them to be successfully utilized to support bone cells and grow bone-like mineral.

The in vivo 3D extracellular matrix provides a temporal regulatory environment of chemical cues. Understanding this dynamic environment will be crucial for efficient drug screening, diseases mechanism elucidation, and tissue engineering. Therefore, in vitro 3D cell culture systems with reversible chemical environments are required. To this end, we developed a non-cytotoxic agarose-desthiobiotin hydrogel to sequester streptavidin biomolecule conjugates (KD 10-11 M), which can then be displaced by the addition of biotin (KD 10-15 M). Streptavidin biomolecule conjugates were simultaneously and sequentially immobilized by changing media components. The time required for biochemical environment exchange was minimized by increasing the surface area to volume ratios and pore size of the hydrogels. We temporally controlled the cell adhesive properties of hydrogels with RGD modified streptavidin to influence endothelial cell tube formation. / Thesis / Master of Science (MSc)

Corneal diseases and insults can result in opacification of the cornea and ultimately lead to blindness. Treatment options for patients are limited due to limited donor availability and the fact that many patients are not eligible for certain treatments due to the nature of their condition. When conventional treatment options are not beneficial for a patient, artificial corneal replacement is necessary. Current artificial replacements induce epithelial downgrowth, where the remaining host corneal cells grow underneath the replacement ultimately leading to implant extrusion. Therefore, surface modification of these synthetic materials is necessary in order to allow proper epithelialization on the surface. This work focuses on the creation of a novel corneal scaffold consisting of poly(2-hydroxyethyl methacrylate) (pHEMA) which is surface modified by 3-(acrylamido)phenylboronic acid (APBA), a molecule known to have cell-binding properties through its ability to bind sugars found throughout the cell membrane. Surfaces were modified using two different polymerization techniques: conventional free radical polymerization (CFRP) and a controlled polymerization technique known as atom transfer radical polymerization (ATRP). It was hypothesized that ATRP would yield more uniform APBA brushes than the conventional method, and therefore create a more efficient cell-binding surface than the conventional method. Following each modification, the surface chemical composition of the materials was confirmed by ATR-FTIR, XPS and surface wettability measurements. Once prepared, NIH 3T3 mouse embryo fibroblasts were seeded onto the surfaces and cell viability was assessed through an MTT assay. The results revealed no cell viability on the APBA-modified surfaces, with surface hydrophobicity, grafting density and surface toxicity (for surfaces modified through ATRP) contributing to the lack of cell attachment. / Thesis / Master of Applied Science (MASc)

Hydrogels are defined as swellable polymer networks with the mechanical, interfacial, and physical properties similar to native tissues in the body. Nanocomposite hydrogels, defined as hydrogels that either entirely consist of or have embedded nanoparticle phases, have been shown to further expand the range of properties achievable with hydrogels and be suitable in many applications such as building tissue scaffolds. In particular, nanocomposite phases that can be eroded offer interesting potential to construct nanoscale voids that can be made in the gel that may be highly beneficial for applications in drug delivery, bioseparations, and tissue engineering. In this thesis, two methods of incorporating starch nanoparticles (SNPs) into a ultraviolet (UV)-cured poly(ethylene glycol methacrylate) (POEGMA) matrix are described. In the first method, the SNPs were physically entrapped during the curing of the gels. An investigation of the effect of fabrication parameters such as monomer ratios, crosslinker amounts, and entrapped SNP concentration on swelling and shear storage modulus (G’) was performed. Enzymatic degradation of the nanophase was also observed to be possible upon amylase treatment, and the resulting internal morphology was confirmed to have increased internal porosity based on a methylene blue uptake experiment. In the second method, chemically functionalized SNPs were used as the exclusive crosslinker to create the POEGMA network. The swelling and mechanical performance of the SNP-crosslinked hydrogels were investigated and compared to the entrapped SNP gels. A preliminary study of the consequences of degradation of a naturally occurring crosslinker to the enzyme α-amylase was also performed. The combination of cytocompatible components and potential for internal porosity control make gels an interesting platform for tissue scaffolding and bioseparation applications. / Thesis / Master of Applied Science (MASc)