This study investigated a CNF/dendrimer hydrogel and how different concentrations of the carboxylated CNF and bis-MPA ammonium dendrimer affected the hydrogels’ rheological properties. A third generation bis-MPA ammonium dendrimer was diffused into a dispersion of carboxylated cellulose nanofibrils. The CNF was carboxylated by TEMPO-oxidation and phosphate buffer deprotonating the carboxylic group. The ammonium dendrimers are cationic and, when added to the dispersion, act as a salt together with the CNF-carboxy anion creating a cationic dendrimer salt bridge. These will serve as physical crosslinks, and a CNF/dendrimer network is formed; the structure and the absorbed water make a hydrogel. Amplitude strain sweeps were performed with a rheometer to determine the gels' elastic capabilities in terms of storage modulus, G’ and loss modulus, G” as the function of the shear stress. The result shows that a higher concentration of both CNF dispersion and dendrimer yielded a higher value of the storage modulus and a lower critical strain, meaning that the hydrogel becomes firmer and less elastic. / I denna studie undersöktes en CNF/dendrimer hydrogel och hur olika koncentrationer av den karboxylerade CNF och bis-MPA ammonium dendrimer påverkar hydrogelens reologiska egenskaper. En tredje generations bis-MPA ammonium dendrimer läts diffusera i en dispersion av karboxylerade cellulosa nanofibriller (CNF). CNF karboxylerades via TEMPO-oxidation, varefter en fosfatbuffer adderades för att skapa en anjon. Dendrimerens ammoniumgrupper är katjoner och då den adderas till dispersionen kommer den agera som ett salt tillsammans med CNF-karboxyanjonen vilket skapar en katjonisk dendrimersaltbrygga. Denna agerar som en fysisk tvärbindning och skapar ett nätverk av CNF och dendrimer. Nätverket skapar tillsammans med det absorberade vattnet en hydrogel.  En amplitude strain sweep utfördes för att bestämma gelernas viskoelastiska förmåga, från mätningarna fås elasticitetsmodulen, G’ och den viskösa modulen, G’’ som funktioner av skjuvningen. Resultatet visar att en högre koncentration av CNF-dispersionen och dendrimeren leder till ett högre värde på elasticitetsmodulen samt ett lägre värde för den kritiska skjuvningen. Detta innebär att hydrogelen blir hårdare och mindre elastisk.

There is a need for a minimally invasive delivery method to enable cell therapies to combat peripheral artery occlusive disease (PAOD) in end stage patients. Myoblasts show promise as a cell mediated therapy but warrant an improved delivery method to increase cell retention in the region of interest because of their adherent nature, relative to previously used BM-MNC’s that are non-adherent. Contemporary issues with achieving successful cell therapies of vasculature can be mainly characterized by the lack of clinical translation from promising animal studies and absence of cell delivery scaffolding. Naturally, polymers have been widely experimented with as grafts to both culture and implant cells into tissue with recognizable success due to their analogous physical properties, such as stiffness, hydrophilicity, & surface energy, that mimic tissue conditions. Polymers having similar mechanical properties to anatomical structures are conducive to cell integration & retention, making polymers an effective biomaterial choice as a cell delivery vehicle. This thesis will evaluate the application of N-isopropylacrylamide (NIPAM) based copolymers as a biomaterial scaffold for myoblast delivery, as it is one of the most widely used biocompatible polymers with thermoreversible properties that is non-toxic and has manipulatable mechanical properties. We hypothesized that fluctuations in polymer construct stiffness, surface energy, and water retention affect myoblast proliferation & viability within the cell delivery vehicle. After measuring the physical properties and cellular proliferation in for each polymer composition, the goal of this thesis was to establish a statistical model to characterize the effect of polymer material properties on myoblast behavior and create a predictive model to optimize further iterations of NIPAM-based copolymers for cell delivery.

The purpose of this lab was to investigate the printability of cellulose nanofiber/carbon nanotubes, their functions as actuators, and to compare these properties with MXene/nano cellulose gels. Data on MXene/nano cellulose gel was obtained from previous research made by Hamedi labs. Data on carbon nanotubes were collected through experiments evaluating different concentrations and sonication times to yield a gel with high conductivity and viscosity. While it was concluded that both gels could be printed into 2D or 3D shapes, the latter failed to maintain its structure over time due to issues with drying. However, it was found that only 2D MXene/CNF could be used as a reversible actuator. / Syftet med laborationen var att undersöka 3D skrivningsförmågan för nanocellulosa/ kolnanorör samt samt deras förmåga att fungera att svälla elektroniskt. Vidare jämfördes dessa egenskaper med MXene/nanocellulosageler. Data på MXene/nanocellulosa insamlades från tidigare experiment gjorda av Hamedi labs. Data på kolnanorör insamlades genom en rad experiment, vilka utvärderade olika koncentrationer och sonikeringstider för att producera geler med hög konduktivitet och viskositet. Slutsatsen blev att båda gelerna kunde 3D printas, men endast MXene/nanocellulosageler kunde användas för elektronisk svällning och avsvällning. Inga geler kunde göras till 3D strukturer.

Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor. Even after patients undergo maximum and safe surgical resection followed by adjuvant chemotherapy and radiation therapy, residual GBM cells form secondary tumors which lead to poor survival times and prognoses for patients. This tumor recurrence can be attributed to the inherent GBM heterogeneity that makes it difficult to eradicate the therapy-resistant and tumorigenic subpopulation of GBM cells with stem cell-like properties, referred to as glioma stem cells (GSCs). Additionally, the migratory nature of GBM/GSCs enable them to invade into the healthy brain parenchyma beyond the resection cavity to generate new tumors. In an effort to address these challenges of GBM recurrence, this research aimed to develop a biomaterials-based approach to attract, capture, and eradicate GBM cells and GSCs with chemical and physical stimuli. Specifically, it is proposed that after surgical removal of the primary GBM tumor mass, an injectable hydrogel can be dispensed into the resection cavity for crosslinking in situ. A combination of chemical and physical cues can then induce the migration of the residual GBM/GSCs into the injectable hydrogel to localize and concentrate the malignant cells prior to non-invasively abating them. In order to develop this proposed treatment, this dissertation focused on 1) characterizing and optimizing the thiol-Michael addition injectable hydrogel, 2) attracting and entrapping GBM/GSCs into the hydrogel with CXCL12-mediated chemotaxis, and 3) assessing the feasibility of utilizing histotripsy to mechanically and non-invasively ablate cells entrapped in the hydrogel. The results revealed that hydrogel formulations comprising 0.175 M NaHCO3(aq) and 50 wt% water content were the most optimal for physical, chemical, and biological compatibility with the GBM microenvironment on the basis of their swelling characteristics, sufficiently crosslinked polymer networks, degradation rates, viscoelastic properties, and interactions with normal human astrocytes. Loading the hydrogel with 5 µg/mL of CXCL12 was optimal for the slow, sustained release of the chemokine payload. A dual layer hydrogel platform demonstrated in vitro that the resulting chemotactic gradient induced the invasion of GBM cells and GSCs from the extracellular matrix and into the synthetic hydrogel with ameboid migration and myosin IIA activation. This injectable hydrogel also demonstrated direct therapeutic benefits by passively eradicating entrapped GBM cells through matrix diffusion limitations as well as decreasing the GBM malignancy and GSC stemness upon cancer cell-hydrogel interactions. Research findings revealed the hydrogels can be synthesized under clinically relevant conditions mimicking GBM resection in vitro, and hydrogels were distinguishable with ultrasound imaging. Furthermore, the synthetic hydrogel was acoustically active to generate a stable cavitation bubble cloud with histotripsy treatment for ablation of entrapped red blood cells with well-defined, uniform lesion areas. Overall, the results from this research demonstrate this injectable hydrogel is a promising platform to attract and entrap malignant GBM/GSCs for subsequent eradication with chemical and physical stimuli. Further development of this platform, such as by integrating electric cues for electrotaxis-directed cell migration, may help to improve the cancer cell trapping capabilities and thereby mitigate GBM tumor recurrences in patients. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is the deadliest type of primary brain cancer. Upon GBM diagnosis, patients first undergo surgery to remove the tumor from the brain. After waiting several weeks for the wound healing process due to surgery, patients are administered chemotherapy with drugs and radiation therapy to eradicate any remaining GBM cells. Even after undergoing these combinatorial treatments, the cancer returns and leads to median survival times of only 15 months in 90% of patients. Complete GBM eradication is difficult, since the cancer cells can migrate into healthy brain tissue beyond the original tumor site. Additionally, GBM is highly heterogenous and composed of different cell types that can resist chemotherapy and radiation therapy, which lead to secondary tumors and cancer relapse. To address these challenges, this dissertation aimed to develop a polymer-based material (specifically a hydrogel) that can attract, entrap, and localize the GBM cells into the material to subsequently eradicate them with chemical and physical signals. This hydrogel platform would have important clinical implications, as it can potentially be dispensed into the empty cavity after surgical removal of the tumor in the brain. The hydrogel can then be harnessed to attract residual GBM cells for directed migration into the hydrogel to concentrate and localize the cancer cells for their subsequent destruction with a non-invasive technology. In order to develop this proposed treatment, this dissertation investigated the following three aims: 1) to study and optimize the injectable hydrogel for chemical, physical, and biological compatibility with the GBM therapy; 2) to utilize chemical signals to attract and entrap the GBM cells into the hydrogel; and 3) to apply focused ultrasound with high amplitude, short duration negative pressure pulses to mechanically fractionate and destroy the cells entrapped in the hydrogel. The results revealed that the hydrogel comprising 0.175 M NaHCO3(aq) and 50 wt% water content was the most optimal formulation. CXCL12 chemokine proteins loaded into the hydrogel at 5 µg/mL released slowly from the hydrogel to generate a chemical gradient and thereby attract GBM cells to promote their invasion into the hydrogel matrix. The hydrogel was demonstrated to respond well to focused ultrasound treatment, which was capable of mechanically fractionating and destroying red blood cells in the hydrogel uniformly. Overall, the results from this research provide support that this hydrogel platform can attract, entrap, and eradicate GBM cells with chemical and physical stimuli. Hence, further improvement of this platform and implementation of this novel GBM treatment may in the future help minimize GBM cancer relapse in patients who undergo conventional therapies, thereby extending their survival times.

Hydrogel membranes with different charge properties, positively charged 2-(N, N-Dimethylamino)ethyl methacrylate-co-butyl acrylate (DMEMA-co-BuA), neutral 2-hydroxyethyl methacrylate-co-butyl acrylate (HEMA-co-BuA), and negatively charged acrylic acid-co-butyl acrylate (AA-BuA) were designed and synthesized for investigation of the transport mechanism of iontophoresis. The hydrogels were characterized by determination of the equilibrium hydration and dimensional change. To study the different crucial factors influencing iontophoretic transport, three model compounds, positively charged phenylpropanolamine (PPA), zwitterionic phenylalanine (Phe), and negatively charged 3-phenylpropionic acid (3-PPA) were selected. These compounds have similar structure and molecular weight but different charge properties. Three transport conditions, passive diffusion, anodal and cathodal iontophoretic transport, were used in this dissertation. A novel parameter, E v , was developed to quantitatively evaluate the enhancement of transport due to the electro-osmotic flux. E v values were obtained by comparing the enhancement/hindrance factor (E-value) through charged membrane to that through neutral membrane with comparable hydration. The model compounds, hydrogel membranes, and transport conditions used in this study made the iontophoretic transport a complicated system with twenty-seven possible different combinations. The E v values can be used to determine the electro-osmotic effect under various iontophoretic conditions with different permeants. The results showed that E v values of permeants with similar structure and molecular weight are robust to permeant charge. Passive diffusion and anodal/cathodal iontophoretic transport of PPA through HEMA-co-BuA and AA-co-BuA of different compositions were conducted. Linear relationships were established between the reciprocal of hydration and the logarithm of flux, E-value, and E v value. It is found that iontophoretic transport of drug through hydrogels is governed by free volume theory. To explore the pH effect on iontophoretic transport through hydrogel, the transport behaviors of Phe through HEMA-co-BuA under three transport conditions in the pH range of 1.5 to 11.2 were investigated. A mathematical model was developed to describe the effect of pH on iontophoretic transport. This model has successfully predicted the total permeability of Phe at various pH values. The distribution and charge of species depended on pH and thus affected iontophoretic transport. The passive, anodal and cathodal transport behaviors of 3-PPA through negatively charged human skin were in agreement with those through the negatively charged hydrogel membrane AA-co-BuA (60:40). It was shown that the negatively charged hydrogel membrane could be used as a model membrane to study transport behavior through human skin.

Yes / Articular cartilage is a vascular tissue with limited repair capabilities, leaving an afflicted person in extreme pain. The tissue experiences numerous forces throughout its lifetime. This study focuses on development of a novel hydrogel composed of chitosan and β-glycerophosphate for articular cartilage repair. The aim of this study was to investigate the mechanical properties and swelling behaviour of a novel hydrogel composed of chitosan and β-glycerophosphate for cartilage repair. The mechanical properties were measured for compression forces. Mach-1 mechanical testing system was used to obtain storage and loss modulus for each hydrogel sample to achieve viscoelastic properties of fabricated hydrogels. Two swelling tests were carried out to compare water retaining capabilities of the samples. The hydrogel samples were made of five different concentrations of β-glycerophosphate cross-linked with chitosan. Each sample with different β-glycerophosphate concentration underwent sinusoidal compression forces at three different frequencies -0.1Hz, 0.316Hz and 1Hz. The result of mechanical testing was obtained as storage and loss modulus. Storage modulus represents the elastic component and loss modulus represents the viscosity of the samples. The results obtained for 1Hz were of interest because the knee experiences frequency of 1Hz during walking.

Filled hydrogel particles consisting of emulsified oil droplets encapsulated within a hydrogel matrix were fabricated based on the phase separation of proteins and polysaccharides through aggregative and segregative mechanisms. A 3% (wt/wt) pectin and 3% (wt/wt) caseinate mixture at pH 7 separated into an upper pectin-rich phase and a lower casein-rich phase. Casein-coated lipid droplets added to this mixture partitioned into the lower casein-rich phase. When shear was applied, an oil-in-water-in-water (O/W1/W2) emulsion consisting of oil droplets (O) contained within a casein-rich dispersed phase (W1) suspended in a pectin-rich continuous phase (W2) was formed. Acidification from pH 7 to 5 promoted adsorption of pectin onto casein-rich W1 droplets, forming filled hydrogel particles. Particles were then cross-linked using transglutaminase. Particles were assessed for stability to changes in pH, increasing levels of salts (sodium chloride and calcium chloride), and susceptibility to lipid oxidation. Both cross-linked and not cross-linked particles were stable at low pH (pH 2-5). At high pH, cross-linked particles maintained their integrity while not cross-linked particles disintegrated. Particles were stable to sodium chloride (0-500 mM). Calcium chloride levels above 4 mM resulted in system gelation. The rate of lipid oxidation for 1% (vol/vol) fish oil encapsulated within filled hydrogel particles was compared to that of oil-in-water emulsions stabilized by either Tween 20 or casein. Emulsions stabilized by Tween 20 oxidized faster than either filled hydrogel particles or casein stabilized emulsions, while filled hydrogel particles and casein stabilized emulsions showed similar oxidation rates. Using an in-vitro digestion model, the digestion of lipid encapsulated within filled hydrogel particles was compared to that of a casein stabilized oil-in-water emulsion. Results showed similar rates of digestion for both hydrogel and emulsion samples. Attempts to fabricate particles using free oil (rather than emulsified oil) were unsuccessful and resulted in the formation of large non-encapsulated oil droplets (d ~10 μm). By controlling particle concentrations of biopolymer, water, and oil, it was possible to fabricate particles that were highly resistant to gravitational separation which was attributed to the equivalent density of the continuous and particle phases. Results highlight the potential applications and versatility of this delivery system.

Damage to our tissues occurs daily and must be repaired by the body in a timely manner in order to prevent infection and restore tissue integrity. Many cell types are involved in the healing process, but it is the cells of the stroma that are largely responsible for rebuilding fibrous tissue, which provides structure and support for all other cell types during healing. This dissertation focuses on stromal tissue repair, the rebuilding of fibrous tissue by fibroblasts following injury. Specifically, I focus on 1) models to study wound healing in vitro, and the specific biological processes of healing that each model captures, 2) the response of engineered stromal microtissues to different methods of injury, namely laceration and laser ablation, and the subsequent clearance and rebuilding of the extracellular matrix by fibroblasts, and 3) how different types of stromal cells and extracellular matrix proteins contribute to tissue repair in vitro.

For a lay summary of the thesis presented in a 1-minute video format, visit the following link: https://www.youtube.com/watch?v=VhLzt_tEz-s / It is projected that, by 2030, 8% of all adults in the world will have diabetes mellitus and treatment will account for 10% of the total healthcare budget in many countries. Polymeric biomaterial research has led to the design of robust polymer hydrogel capsules to develop curative cell-based therapies for chronic disorders such as diabetes mellitus. Encapsulation of insulin-producing beta cells within synthetic, semi-permeable polymer hydrogels can avoid host immune rejection including fibrotic responses, and thus holds the promise of a long-term curative treatment of this disease. There is a paucity of literature regarding methods available for standardized in vitro screening of synthetic polymer hydrogel capsules to predict host responses in vivo. Thus, the focus of this thesis was to design in vitro assays able to screen for subsequent in vivo fibrotic responses. Two dimensional (‘2D’) (cell attachment to thin film hydrogel coatings) and three dimensional (‘3D’) (cell attachment and protein adsorption to hydrogel capsules) in vitro experiments were designed and tested in an iterative process to assess fibrotic responses to a diverse group of polymer hydrogels. Cell attachment assays included fibroblast (NIH 3T3) and macrophage (RAW 264.7) cell lines, and protein adsorption assays included proteins used to model fibrosis including fibrinogen and lysozyme. For some formulations, in vitro assays were compared with in vivo data on pericapsular cellular overgrowth (PCO) after being implanted into mice. A binomial logistic regression model was designed and validated to assess whether the ‘3D’ in vitro assays correlated with in vivo PCO responses. It was found that the RAW 264.7 cell attachment assay was significantly correlated with PCO outcomes in vivo, demonstrating for the first time a simple, cost-effective, and rapid in vitro cell-based approach to screen and select capsules with lower fibrotic potential to be further tested in animals. / Thesis / Master of Health Sciences (MSc) / In North America, one in eleven adults, or about 415 million people, have diabetes. It is projected that by 2030, around 8% of the world population will be diagnosed with this disease. A common form of treatment is through the frequent injection of insulin, but this is costly, requires multiple daily interventions, and cannot prevent regular excursions from the ideal blood glucose range. Cell-based therapies have a lot of promise in treating several chronic diseases including diabetes. Donor and stem-cell derived islets can be implanted into patients with type 1 diabetes and have been shown to function for over a year, albeit at the price of systematic immune suppression. Alternatively, cells that produce insulin can be placed inside immune-evasive capsules and implanted, potentially providing continuous blood glucose regulation without the need for daily insulin injections. However, this novel form of treatment is limited by the encapsulated cells’ survival once implanted. Cell survival can be affected by the body’s response to a foreign body (the capsule), causing deposition of protein or cells on the capsule surface which can limit the oxygen supply to cells in the capsule and the ability of insulin to leave the capsule in a timely fashion. The goal of this project is to develop assays to screen new capsule formulations. This can advance research by using capsules more readily accepted by the body, leading to a more promising and long-term treatment of diabetes.

Spinal cord injury causes the partial or total loss of the anatomical and functional continuity of the spinal cord tissue, leading to the damage of the organs controlled by nerves that branch off downstream the injury. This thesis analyses the mechanics of two possible treatments based on two different approaches: intraspinal microstimulation (ISMS) and tissue engineering. These two approaches have a common rationale, the delivery of electrical stimuli to the injured spinal cord. In the literature, the feasibility of the electrodes for ISMS is often limited to the analysis of stiffness. The mechanical validation of the device is then focused on the step after the in vivo implantation, considering the interplay with the surrounding tissue. In this work, the mechanical performance of an innovative intraspinal microstimulation device is evaluated thoroughly before the in vivo step, to avoid the waste of material, animals, and time. The study involves the characterisation of the single components (electrodes), prototypes, and possible failure mechanisms. A work on silk fibroin hydrogels for the regeneration of the spinal cord is also presented. Silk fibroin is a highly versatile material for biomedical purposes, and thus largely used in tissue engineering. Moreover, it has piezolectric properties subjected to micro and nanostructure. Given the proven benefits of electrical stimulation in the regeneration of the spinal cord after injury, different approaches studied in literature often require the use of external devices to generate electrical stimuli. This thesis aims to study the mechanical properties of silk fibroin hydrogels obtained by applying an electric field to silk fibroin solutions, to investigate the eventual increase of the microstructure orientation and consequent improvement of the piezoelectric effects of fibroin.