Biomedical Engineering - Theses
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ItemMagnetic manipulation of cells to enhance tissue engineering( 2023-03)One of the major detrimental effects of the aging process is the natural atrophy of skeletal muscle tissues, a process where individuals typically experience lower muscle mass, reduced muscle function and, as the problem worsens, compromised personal independence. This problem can become exacerbated due to injuries and diseases such as cancer, where patients may suffer from cachexia, a severe form of atrophy which leads to a 20-70% total volume loss in affected muscles. As it stands now, in vitro tissue engineered muscle fibres are often-times functionally immature, making it difficult to use these fibres as experimental models for drug testing. Part of the problem is that current tissue engineering methods use complex and expensive bioreactor systems to exercise skeletal muscle cells in vitro, systems that are inherently inflexible in terms of their potential applications. This technological shortcoming limits the insights that can be gained about muscle development and its disease states. Therefore, the aim of this thesis was to develop an in vitro magnetic stimulation method that provides a finer, more flexible alternative to typical bioreactors, and to investigate possible applications of this novel stimulation method for C2C12 myoblasts grown in a variety of 2D and 3D environments. To achieve this aim, magnetic chitosan microspheres (1-10 micron diameter) were produced and loaded with 30 nm, polyethylene glycol (PEG)-coated iron oxide nanoparticles (Mag30-CMs). The microspheres were produced at this size to limit cellular uptake prior to stimulation, and the microspheres were further functionalized with an RGD-containing peptide (Mag30-CM-RGDs) to enable external cellular stimulation through key integrin receptors. A magnetic stimulation chamber, optimized using finite element simulations, was 3D-printed and contained a sterile culture plate and two N45-grade neodymium magnets, allowing for the culture of magnetically-labelled C2C12 myoblasts inside a controlled and well-defined static magnetic field. After this system was developed, the same stimulation regime was applied to C2C12 myoblasts grown on a soft (8 kPa) 2D gelatin methacryloyl (GelMA) hydrogel scaffold, in order to determine the efficacy of this technique on softer substrates. Finally, the stimulation regime was then applied to C2C12 myoblasts grown in RGD-functionalized chitosan-gelatin cryogels, with macrostructures consisting of interconnected aligned pores roughly 50-250 micron wide. The maturity of the differentiated myotubes produced in the three environments was characterized based on the qPCR gene expression of key myogenic regulatory factors (MRFs), as well as morphological analysis (fusion index) done via confocal imaging. The magnetic stimulation regime method was modelled to produce piconewton-sized forces directly on the cell surface, resulting in a significantly higher fusion index as well as up-regulated gene expression of key MRFs for stimulated myotubes on hard tissue culture plastic (TCP). Stimulated myotubes had a 5-fold increase in multinucleated myotubes with 4+ nuclei over control, an important indicator of maturity. Significant increases from 2-6 fold were found in the gene expression of mature myosin heavy chain (MHC) genes MHC1, MHC2x, and MHC2a, in addition to a 2-2.3 fold increase in myogenin, a key MRF. These results indicate a promotion of myotube maturity in vitro in response to magnetic stimulation via the application of Mag30-CM-RGDs in a static magnetic field on hard TCP. In comparison, the soft GelMA hydrogels did not see the same benefits from the stimulation regime, producing no significant changes between the stimulated group and control. The 3D porous cryogels, functionalized with RGD-containing peptides, demonstrated some evidence of good cell attachment at early timepoints, but the attachment was not robust enough for the cryogels to serve as a scaffold for this kind of stimulation method, and cells did not survive long enough inside the gels to undergo differentiation. In this thesis, a robust and flexible magnetic stimulation method was produced and investigated in a novel skeletal muscle tissue engineering application. The Mag30-CM-RGDs showed evidence of maintaining cell attachment after 5 days of differentiation, and were able to promote muscle maturity in a 2D setting. This technique is not unique to skeletal muscle cells, and the methods utilized here can be adapted and applied to other types of tissues. As the materials are biocompatible, future experiments can be conducted to determine if these materials can be effective in vivo as well as in vitro.
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ItemTargeting seamless cartilage repair with a bioadhesive implant( 2022)Injury to articular cartilage in the knee can lead to post-traumatic osteoarthritis if untreated, causing debilitating problems later in life. Osteoarthritis impacts an individuals’ mobility, ability to work, and participation in daily activities. The broader impacts of this are significant, with osteoarthritis a leading cause of disability and an economic burden globally. Standard surgical treatments fail to ensure long lasting repair of damaged cartilage, often resulting in fibrotic tissue. The field of tissue engineering has seen a vast amount of research in cartilage regeneration, with few strategies reaching clinical trials. A common theme among failure of tissue engineered implants is their inability to integrate with the native tissue. Cartilage is a deceptively complex tissue despite its lack of innervation or blood supply. Its matrix is dense, heterogeneous and anti-adhesive, containing only a small number of cells and little ability for self-repair. This work seeks to understand if a cell-laden bioadhesive material can improve integration with cartilage, by bonding the regenerative implant to the native tissue. A novel bioadhesive comprised of photocrosslinkable gelatin methacryloyl and a biological enzyme, microbial transglutaminase, is reported. The material’s adhesion to cartilage ex vivo is assessed mechanically and chondrogenesis by human adipose derived stem cells (hADSCs) encapsulated within the material is evaluated. The enzyme significantly improved adhesion to cartilage ex vivo and did not impede the production of cartilage matrix by hADSCs cultured under chondrogenic stimulation conditions. In a preliminary study, the enzyme significantly improved integration with human cartilage explants over time under static culture conditions. The ability of the bioadhesive to support integration with cartilage ex vivo under cyclic compressive loading was then investigated, which is understood to be a first within the literature. No clear advantage of the bioadhesive was observed under loading, with good integration observed histologically in all conditions and a significant ten-fold increase in integration strength over the culture duration. This experimental model in combination with a biphasic finite element model allows future investigation of open questions in the field. Further work could see the bioadhesive material combined with other strategies to improve integration and long-term cartilage regeneration outcomes, providing an essential step towards clinical translation.
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ItemNo Preview AvailableTuning the Bioactivity of Cell-derived Extracellular Matrices through Control of Substrate Properties( 2021)Mesenchymal stromal cells (MSCs) are the subject of thousands of clinical trials for treatment of innumerable human pathologies. However, their widespread clinical use is still hampered by difficulties in retaining their stem cell-like properties during prolonged ex vivo expansion. The use of cell-derived extracellular matrix (ECM) has recently garnered much interest as a culture substrate because ECM significantly improves cell viability in ex vivo culture. However, these biomaterials are themselves produced in an environment which is not physiologically accurate, and therefore, in this study the aim was to optimise bioactivity of cell-derived ECM by controlling substrate properties to more accurately mimic the in vivo niche. MSCs are innately sensitive to substrate stiffness and their surrounding mechanical environment. The first results chapter (Chapter 2) describes investigation into whether manipulation of substrate stiffness increases bioactivity of cell-derived ECM. Polyacrylamide hydrogels with tunable stiffnesses were fabricated, and MSCs induced to deposit ECM on these surfaces. Subsequent culture and behavior of primary MSCs on the ECM was then observed. Primary cells cultured on ECM deposited on soft substrates demonstrated the highest levels of proliferation. On the other hand, there were significantly higher levels of osteogenesis when culturing MSCs on ECM deposited on stiff substrates. These results show that the bioactivity of ECM can be taken even further by controlling mechanical properties of substrates that mimic the cellular microenvironment. In addition to substrate stiffness, surface chemistry was controlled to determine whether this affected ECM bioactivity in a similar fashion, described in Chapter 3. The surface chemistry of glass coverslips was modified with various silanes, introducing amine, carboxylic acid, propyl, and octyl functional groups onto the surfaces, and studied how introduction of these moieties affected the ECM. ECM on these surfaces improved cellular proliferation and adipogenesis. However, the bioactivity of the ECM did not depend significantly on the surface chemistry for the range of chemistries and culture timescales tested. These results allow for greater flexibility in fabricating tissue engineering materials with a variety of surface chemistries for use as ECM scaffolds. Lastly, Chapter 4 describes an investigation to determine whether the beneficial effects observed by tuning substrate stiffness could be scaled up and applied to 3D culture systems. Porous polyacrylamide cryogels were fabricated for the culture of MSCs and subsequent ECM deposition. MSCs cultured in this manner did not demonstrate improved cell behavior. However, these 3D scaffolds were a suitable material for the deposition of cell-derived ECM, providing a future avenue of research for the scaleup and clinical translation of these matrix biomaterials. This dissertation presents findings which demonstrate that the effects of varying substrate stiffness can be combined with use of ECM to increase MSC proliferation and osteogenesis. Altering surface chemistry is also shown to not have a significant effect on dECM bioactivity for the range of functional groups tested. Fabrication of tunable porous cryogels with ~65 um pores also revealed that secretion of ECM by MSCs is not affected sufficiently to result in changes to its bioactivity. These findings contribute to the field by showing that parameters such as substrate stiffness can be manipulated and used with ECM in cell culture to result in improved cell behavior compared to using ECM alone, and also illustrates that strict control of other variables such as surface chemistry and three-dimensionality are not necessary in order to maintain the bioactivity of cell-derived ECM.
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ItemImproving ex-vivo expansion of mesenchymal stromal/stem cells using acellular fetal membranes( 2019)Mesenchymal stromal/stem cells (MSCs) have considerable potential in the fields of cell therapies, tissue engineering, and regenerative medicine. According to clinicaltrials.gov, MSCs are employed in more than 700 registered clinical trials as potential treatments. However, the clinical application of MSCs is limited by their low prevalence in the human body and inefficient methods for large-scale ex-vivo production. During ex-vivo expansion, MSCs experience a vastly different environment compared to their natural microenvironment (i.e. their niche), and these environmental differences are likely to be main drivers for the loss of key MSC properties. In this study, the general aim was to investigate the effect of two main components of the MSC niche on decidua-derived MSCs (DMSCs) from human placenta during ex-vivo expansion; the extracellular matrix (ECM) and extracellular vesicles (EVs). Coatings produced from ECM are promising surfaces for the improved ex-vivo expansion of MSCs. However, identifying a readily available source of ECM to generate these coatings is the bottleneck of this technology. In Chapter 2 of this thesis, ECM coatings derived from decellularised fetal membranes were assessed as suitable substrates for MSC expansion. The fetal membrane’s two main components, the amnion and the chorion, were separated, decellularised and processed further to produce solubilised forms of the decellularised amniotic membrane (s-dAM) and decellularised chorionic membrane (s-dCM). DMSCs were more proliferative, smaller in size (a measure of MSC potency, and exhibited greater adopogenic and osteogenic differentiation capacity when cultured on s-dAM compared to controls. Additionally, long-term culture studies revealed that late passage DMSCs (passage 8) cultured on s-dAM had decreased cell diameter over three passages. These data support the use of s-dAM as a substrate for improved MSC expansion. In addition to the ECM, extracellular vesicles are another important component of the MSC niche. However, the contribution of ECM and EVs has not been explored from an MSC expansion point of view. In Chapter 3, the effect of adding MSC-derived EVs to DMSCs cultured on the ECM coatings described in Chapter 2 was assessed. Addition of EVs to the DMSCs growing on Matrigel improved their attachment. However, regardless of the presence of EVs, DMSCs showed significantly better attachment on s-dAM. Furthermore, addition of EVs to DMSCs growing on s-dAM improved DMSC proliferation, migration and osteogenic capacity. The total antioxidant capacity of DMSCs growing on Matrigel, s-dAM and s-dCM increased, regardless of whether EVs were added to DMSCs or not. These data illustrate the relative contribution of ECM and EVs towards MSC expansion and show that supplementing the MSC culture with EVs can regulate certain MSC properties. In addition to issues of large-scale ex-vivo expansion of MSCs, their clinical application is limited due to their low survival rates and poor engraftment after delivery. A number of factors contribute to these issues including cell damage due to shear stress during injection, leakage of cells from the injection site, and lack of appropriate interactions with surrounding cells and extracellular matrix. In Chapter 4 of this thesis, s-dAM and s-dCM were investigated for their potential to form 3D thermoreversible and injectable hydrogels to be used as a cell delivery carrier for MSCs. At 37ºC, both s-dAM and s-dCM formed gels at concentrations of 4 and 8 mg/mL. DMSCs growing on s-dAM showed improved proliferation, adipogenesis and osteogenesis compared to TCP. Both s-dAM and s-dCM had shear thinning properties, and were therefore injectable. DMSCs embedded in both s-dAM and s-dCM had a viability of ~60% after injection and showed improved proliferation compared to Matrigel. These data support that the ECM from both s-dAM and s-dCM can be processed to produce thermoreversible and injectable hydrogels, and s-dAM hydrogels promote key properties of DMSCs.