Medicine (St Vincent's) - Theses
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ItemEngineering articular cartilage from human infrapatellar fat pad stem cells for transplantation therapy( 2015)Mesenchymal stem cells (MSCs) have shown promise in cartilage tissue engineering due to their unlimited capacity for self-renewal and capability to differentiate into cartilage tissue lineage under certain physiological or experimental conditions. In this thesis, we harvested MSCs from human infrapatellar fat pad tissue (hIPFP) and further fully characterised using flow cytometry. Human IPFP-derived MSCs at passage three (P3) show good homogeneity for MSCs cluster differentiation (CD) markers including CD29, CD44, CD73, CD90, and CD105. Hyaline articular cartilage repair is a significant challenge in orthopaedics and traditional therapeutic options result in inferior outcomes. We believe traditional methods can be improved through applications based on three-dimension (3D) culture systems and tissue engineering strategies. In this thesis, we planned to investigate the chondrogenic potential of hIPFP-derived MSCs, stimulated by TGFβ3 and BMP6, over 7, 14 and 28 day in vitro in 3D pellet culture, a 3D printed chitosan scaffold and a 3D scaffold comprising methacrylated hyaluronic acid and methacrylated gelatin (called HA/GelMA). Therefore, endpoints included histology staining, immunohistochemistry, immunofluorescence, and temporal changes in expression of specific chondrogenic genes using quantitative real-time polymerase chain reaction (qPCR). In vitro 3D pellet culture maintained cells to be in close proximity to each other and promoted cell aggregation that mimics the cellular condensation process within native cartilage tissue. Furthermore, research has shown the potential of 3D biomaterial scaffolds for providing a suitable environment for chondrogenic induction and significantly enhancing the proliferation, differentiation, and chondrocytic extracellular matrix synthesis by MSCs. Collaborators at the Intelligent Polymer Research Institute (IPRI) at the Uiversity of Wollongong have developed extrusion printing for diverse bioengineering projects and this technique has developed for provision of both 3D chitosan scaffolds and 3D hyaluronic acid/biogel scaffolds for this project. The biocompatibility of chitosan and its structural similarity with glycosaminoglycan make it attractive for cartilage tissue engineering. Also, methacrylated HA and gelatin polymers were utilised to produce UV- crosslinkable HA/GelMA scaffold. A cartilage extracellular matrix component, HA, is the main non-sulphated glycosaminoglycan and offers a promise candidate for engineering of cartilage. In all three types of cultures (pellet, chitosan and HA/GelMA), over 14–28 days, clusters of encapsulated chondrocytes formed. Collagen type 2 and proteoglycan production were confirmed using immunohistochemistry and immunoflourescence. Chondrogenic lineage markers including: SRY-related transcription factor (SOX9), collagen type 2 alpha 1 (COL2A1), and aggrecan (ACAN) gene expression increased significantly over the time course. We reported that chitosan and HA/GelMA scaffolds enhance and increase the efficiency of chondrogenesis in our model. Finally, advanced microarray technique was conducted to provide novel informations about overall gene expressions during chondrogenesis across all three cultures. This is the first time that in vitro microarray has been used in the assessment of the chondrogenic differentiation of hIPFP-derived MSCs cultured in 3D pellet and seeded into chitosan and HA/GelMA scaffolds. Microarray gene analysis requires high-end programming for assessment of the test statistics that show whether a particular gene or a set of related genes are highly regulated (up- or down-regulated). Another challenge is to select a ‘ranking of expressed genes’ that may be relevant to a particular set of experimental conditions or of particular interest from a biological perspective (e.g. a particular metabolic pathway or a set of apoptotic genes). Therefore, we have successfully demonstrated in vitro production of hyaline-like cartilage from infrapatellar fat pad (IPFP)-derived MSCs in 3D culture. Microarray has provided novel informations concerning genes involved in chondrogenesis of hIPFP- derived MSCs and our approach offers a viable strategy for generating clinically relevant cartilage for therapeutic use.
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ItemCharacterisation, optimization and transplantation of a tissue engineered cardiac muscle flap( 2014)The field of tissue engineering presents a new means of generating tissues for reconstruction. Engineering functional myocardium de novo can potentially address the current challenges faced in the field of heart transplantation and congenital cardiac abnormalities. However, heart tissue is metabolically demanding, and therefore highly susceptible to ischaemia. As the main strategies to engineer myocardium rely on assembling cardiomyocytes in vitro using scaffolds (e.g. polymer based or hydrogel based) or in a form of scaffoldless cell-sheets, its survival through implantation in vivo relies on neovascularisation from the recipient bed. This presents a major hurdle for cardiac tissue engineers, as these ‘cardiac grafts’ are unlikely to survive, especially, in the harsh environment of ischaemic tissue post- myocardial infarction. Publications quantifying the survival of cardiac tissues after implantation in vivo are scarce but conservative estimates suggest that less than one-tenth of such grafted cardiac tissue will survive. Using an in vivo vascularisation approach at our laboratory, by placing a microsurgically fabricated arteriovenous loop into a polycarbonate chamber (AV loop tissue engineering chamber), cardiomyocytes suspended in a hydrogel-based scaffold assembled in vivo into an ‘engineered vascularised cardiac muscle flap’ that is potentially transplantable. While this concept seemed an attractive solution to the problem facing cardiac tissue engineering, several questions have yet to be investigated: 1. Will the tissue engineered cardiac muscle flap suffer minimal tissue loss, after transplantation? 2. Following transplantation to the epicardium, will the engineered cardiac muscle tissue integrate with the myocardium? In this thesis, a series of experiments were performed to answer these questions. The information obtained in the studies can be summarised as follows: A. Seeding syngeneic neonatal rat cardiomyocytes into the AV loop tissue engineering chamber with MatrigelTM as a scaffold, it is possible to generate a contractile cardiac muscle flap in Sprague Dawley rats. Immunohistological examination showed no signs of acute rejection. This implies the AV loop approach may be suitable when autologous cell sources are available for implantation. B. In a dose-response experiment, seeding 6 million cardiomyocytes per chamber was found to produce a small variance and a significant mean volume. This has important implications for the outcome of histomorphometric analysis of the flap. It was also observed that the cardiac tissue volume generated seemed to demonstrate a ‘dose-response trend’, that was not seen in other existing cardiac tissue engineered approaches. C. When investigating two isoforms of an enzyme system, NADPH oxidase, in hope of boosting angiogenesis to generate robust cardiac tissues, it was found that the angiogenic environment in the tissue engineering chamber was too complex to be altered by simply targeting a single factor. D. Subjecting the tissue engineered cardiac muscle flap to ischaemia time and conditions similar to that seen in the heterotopic rat heart model, the cardiac muscle flap did not show any quantitative loss or morphological changes when transplanted to an ectopic site. E. The small size of the Sprague Dawley rats and its anatomical features did not allow the cardiac muscle flap to be transplanted in an autologous fashion to the heart. A novel model using two syngeneic adult rats, allowed transplantation of the cardiac muscle flap based on a long pedicle from one rat to the other’s heart. The method was feasible and reproducible. Histological examination of the transplanted flap showed connective tissue integration of the flap with the host’s heart, however, the flap’s cardiac tissue remained separated from the myocardium by some collagenous tissue. In summary, a syngeneic cardiac muscle flap was generated in the AV loop tissue engineering chamber and the concept of a transplantable vascularised cardiac muscle flap was demonstrated. The novel allogeneic transplant model may have application as a platform for testing functionality of various cardiomyogenic stem cell sources. While much is there to overcome, this is a step forward in translating this approach from the bench to bedside.
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ItemTrophic role of adipose-derived adult stem cells to support tissue engineering( 2012)Tissue engineering has held much promise for patients suffering from irreversibly damaged organs/tissues who are in desperate need for organ transplantation. Despite technological advances in the biochemical engineering of scaffolds to permit efficient vessel growth during tissue development, vascularisation still represents a major limiting factor in the generation of tissues large enough for clinical applications. Human mesenchymal stem cells (MSC) were originally proposed for stem cell therapies in regenerative medicine due to their propensity to differentiate into specific cell types. However, MSC were found to be more supportive of engineering functional tissue constructs through secretion of a spectrum of growth factors and cytokines, termed paracrine factors, which are angiogenic and cytoprotective. In this thesis, it is aimed to compare the paracrine profile of various MSC populations and determine changes in the expression profile when the cells are induced to differentiate down a specific lineage. In addition, an efficient regulatory method that would enhance the paracrine activity of ASC was investigated and translated into an in vivo animal model. In determining the optimal MSC population for promoting angiogenesis through paracrine activity, human MSC isolated form bone marrow (BMSC), adipose tissue (ASC) and dermal sheath (DSC) or papilla (DPC) of hair follicles were compared. While expression of selected paracrine factors, including SDF-1, VEGF-A, VEGF-C, bFGF, HGF, NGF and ANG, exhibited minor differences within MSC populations examined, ASC expressed the highest levels of IGF-1, VEGF-D and IL-8. Furthermore, ASC-derived conditioned medium (ASCCM) induced the strongest response in proliferation, migration and tube formation of human microvascular endothelial cells (HMEC) in vitro. ASC were therefore suggested as a suitable MSC population for angiogenesis-related applications, as not only can a large number of ASC be generated with minimally invasive isolation procedures, ASCCM also demonstrate an advanced capacity to support angiogenesis in vitro. It is further demonstrated in this thesis, that the unique paracrine factor profile of ASC is altered when the cells commit to specific cell lineages, such as adipogenic and osteogenic. Progress through differentiation significantly diminished the expression of potent angiogenic factors, including VEGF-A and bFGF, so it was hypothesised that this may impact negatively on their angiogenic paracrine activity. Therefore, the paracrine activity of ASC is suggested to be a unique characteristic present only when the cells are multipotent. In addition, it is likely that the beneficial angiogenic activity of ASC paracrine factors in regenerative therapies is associated with their “stemness”, and that maintenance of ASC stemness during tissue formation may benefit the outcome through enhanced angiogenesis in vivo. Lastly, hypoxia was examined as an efficient method to enhance the paracrine factor production of ASC, where both VEGF-A and ANG were significantly increased when cells were subjected to conditioning by hypoxia. The angiogenicity of ASCCM was confirmed by implanting polyvinyl alcohol sponge subcutaneously in mice, where the ability of concentrated ASCCM to promote vascularisation in animal implanted sponges was determined by immunohistochemical staining of the endothelial cell specific marker CD31. Moreover, the ASCCM collected from hypoxia-conditioned cells exhibited enhanced vessel infiltration in sponges, which was diminished by neutralising antibodies against VEGF-A and ANG. The model demonstrated that the increased production of VEGF-A and ANG in hypoxia-conditioned ASC in vitro translated to an in vivo effect with biological significance. Collectively, these results illustrated the potential for utilisation of ASCCM alone for angiogenesis-related applications in vivo. In summary, the data presented in this thesis presents ASC as a useful MSC population for enhancing angiogenesis because of their optimal paracrine factor profile. While the paracrine factor expression is unique to multipotent ASC, the angiogenicity of its paracrine activity can be further enhanced through in vitro hypoxia conditioning. The increased production of VEGF-A and ANG contributed to the observed enhancement of blood vessel infiltration in the in vivo sponge implant and provided evidence of the paracrine activity of ASC. The results demonstrated in this thesis therefore suggest the potential of ASCCM as a suitable agent for induction of angiogenesis, which may be incorporated within scaffold materials to increase vascularisation efficiency of the tissue engineering construct.
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ItemPreconditioning stem cells for cardiac tissue engineering( 2010)Stem cells represent a promising tool for cell and tissue therapies due to their propensity to differentiate as well as their anti-apoptotic, pro-angiogenic and tissue remodeling properties. In practice however, the successful implementation of these cells into cell and tissue therapies for treating the complications of myocardial infarction has been limited by poor cell survival following implantation. Preconditioning stem cells prior to implantation to the ischaemic myocardium has been shown to improve implanted cell survival as well as improve functional outcomes. Preconditioning cells for tissue engineering however, remains largely unexplored. In this work, I aimed to explore how adipose-derived stem cells (ASC) can be protected against the hypoxic and ischaemic environments that occur in tissue engineering. I also aimed to determine whether preconditioning improves the survival of implanted stem cells in an in vivo tissue engineering environment. In establishing a model of ASC death in vitro, we found that ASC were resistant to severe hypoxia for up to 72 h but not to ischaemia. After just 24 h ischaemia, ASC morphology was altered consistent with cellular apoptosis. Concomitant with altered morphology, ischaemia significantly decreased cell viability compared with normoxia as measured by cell counting and MTT-based absorbance assays. Furthermore, compared with normoxia, ischaemia significantly increased cellular injury and apoptosis as measured by lactate dehydrogenase release into the medium and caspase 3/7 activity respectively. In investigating the susceptibility of other cell types important for cardiac tissue engineering to hypoxia or ischaemia-induced cell death, we found that endothelial cells and cardiomyocyte-like cells derived from ASC were much more susceptible to cellular injury and apoptosis during severe hypoxia alone. Our exploration of protection protocols showed that preconditioning ASC with hypoxia for 24 h protected them against ischaemia-induced cell death in vitro. Preconditioned ASC expressed significantly increased levels of hypoxia inducible factor-1α (HIF-1α) protein and its downstream target gene, vascular endothelial growth factor-A (VEGF-A) mRNA. This corresponded with a significant increase in hypoxia-mediated VEGF-A secretion into the culture medium. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned cells. The protective effects of hypoxic preconditioning were abolished by a neutralising antibody against VEGF-A and the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 demonstrating the importance of VEGF and Akt in ASC survival. Interestingly, the protective paracrine effects of ASC were also potentiated by hypoxic preconditioning. We found that conditioned medium from hypoxic preconditioned ASC significantly decreased hypoxia-induced endothelial cell death compared with conditioned medium from non-preconditioned ASC. Hypoxic preconditioned ASC also promoted angiogenesis to a greater extent than non-preconditioned ASC. The addition of VEGF to conditioned medium from non-preconditioned ASC showed that this effect was mediated through the increase in ASC VEGF-A secretion under hypoxic conditions. Neither autocrine preconditioning protocols nor the protective paracrine effects of hypoxic preconditioned ASC on cardiomyogenic ASC were investigated however due to unreliable differentiation protocols. Finally, we investigated ASC death following implantation into an in vivo tissue engineering chamber. Immunohistochemisty revealed that ASC implanted into an in vivo tissue engineering chamber were able to survive the first two days following implantation. ASC nuclear morphology was regular and did not display any hallmarks of apoptosis such as chromatin condensation. Furthermore, we were unable to detect any cleaved caspase-3-positive ASC within the constructs at this time point suggesting that ASC were not apoptotic and had survived implantation through to two days. ASC were distributed throughout the chamber, remained in the growth factor-reduced Matrigel™, and tended to aggregate into clusters. Moreover, HIF-1α was present in these cells suggesting that within this environment, ASC were hypoxic and that HIF-1α may have contributed to their survival in these conditions. In collating this data, it appears that ASC are able to survive severe hypoxia both in vitro and in vivo, at least in part via a HIF-1α/VEGF-A/PI3K-Akt-mediated mechanism, and exert protective effects, which were potentiated following hypoxic preconditioning of ASC, on other cell types. Therefore, rather than requiring protection in in vivo tissue engineering environments themselves, it appears that hypoxic ASC may have an important role in supporting the survival of endothelial cells and possibly stem cell-derived cardiomyocytes in such environments for support and stabilisation of tissue growth.