Surgery (St Vincent's) - Theses
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ItemDeveloping liver specific vasculature to support the growth of liver progenitor cells for liver tissue engineering( 2015)Tissue engineering is the combination of organ/tissue specific cells in matrices or scaffolds to grow new tissues. Tissue engineering and the related area of cell therapy hold much promise for the repair, replacement and regeneration of organs and tissues damaged by disease and trauma. To date, liver tissue replacement research has focused on cell therapies – particularly hepatocyte transplantation into the diseased liver which specifically aims to reduce the need for liver transplantation, and aims to treat the myriad of end stage liver diseases and metabolic disorders. Despite the technological advances, a major limitation to the clinical success of hepatocyte cell therapy and other liver tissue engineering strategies is the inability to generate a vascular supply capable of supporting the engineering of large, three-dimensional (3D) tissues and organs. The liver sinusoidal endothelial cell (LSEC) makes up the liver specific microvascular network (sinusoids) and plays an integral role in liver development, liver homeostasis and liver regeneration. Vascularisation itself is rarely addressed in liver tissue engineering; let alone the incorporation of LSECs in liver constructs. In this study murine LSECs were used to construct liver specific blood vessels and were identified by their surface markers (LYVE1+/ CD31-), as opposed to capillary endothelial cells (LYVE1-/ CD31+), and lymphatics (LYVE1+/ CD31+). LSECs were tested in vitro and in vivo with and without the addition of murine liver progenitor cells (LPCs). Liver progenitor cells are a native liver progenitor cell, capable of differentiating into both hepatocytes and cholangiocytes. Whilst hepatocytes are the gold standard for liver tissue engineering, LPCs offer an alternative, rarely investigated source of hepatocytes for tissue engineering. LPCs are responsible for liver regeneration during end stage liver disease, when hepatocyte proliferation has been impaired. As LSECs and LPCs play important roles in liver regeneration, both cell types were investigated for their possible applications in liver tissue engineering. LSECs and LPCs were cultured as 3-D multicellular spheroids of one cell type-termed homospheres, or co-cultured together as heterospheres in vitro, for subsequent in vivo implantation in a vascularized tissue engineering chamber. This thesis demonstrates that LSECs and LPCs are capable of forming homogeneous homospheres, and heterogeneous spheroids of co-cultured heterospheres. Furthermore, LSECs form vascular structures in vitro when cultured as homospheres heterospheres. The ability to generate vascular structures through co-culturing with endothelial cells in vitro is termed pre-vascularization, and is currently at the forefront of vascular tissue engineering. The thesis also demonstrates that LSECs were capable of integrating into native vasculature when implanted in vivo to form a liver specific vasculature at an ectopic site in healthy SCID mice. The liver specific vasculature was identifiable as LYVE1+/ CD31- and through the detection of DiI labeled LSECs. Implantation of LSECs also increased the generation of native neo-vasculature (LYVE1-/CD31+)- this was significant when the matrix Matrigel was used in the tissue engineering chamber. However, the generation of liver specific vasculature and increased native vasculature did not support significant survival of LPCs. Whilst the results indicate that heterospheres of LSECs and LPCs improve the survival and spontaneous hepatocyte differentiation of LPCs, the overall survival was inconsistent throughout the in vivo experimental groups in SCID mice. Finally, LPC spheroids were implanted in a mouse model of the metabolic disorder methylmalonic aciduria (MMA). LSECs were not used in the final study; as LPC survival was inconsistent and low regardless of the presence/absence of LSECs. Instead, the total number of LPCs implanted was increased 4 fold. Again, survival of LPCs was low and appeared to elicit an immune reaction in MMA mice. Ultimately, the final study did not demonstrate any functional disease reversal, due to poor LPC survival. This is the first liver tissue engineering study that has used LSECs with LPCs. Isolated LSECs demonstrated significant promise in their ability to generate a liver-specific vasculature in vivo for liver tissue engineering, however the use of LPCs presented a number of issues, which require further investigation – the most significant of which was their inconsistent and generally poor survival in vivo.
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ItemThromboembolism and neoadjuvant chemoradiation for rectal cancer( 2015)Thromboembolism (TE) is one of the leading causes of morbidity and mortality in cancer, is an independent predictor of reduced survival, and the overall rate of TE in cancer patients is increasing. Assessing the TE risk of an individual patient at a given time point and the benefit of thromboprophylaxis (TP) can be complex, involving widely variable TE rates according to tumour related factors (such as cancer subtype and disease stage), as well as patient and treatment related factors. Some, such as surgery and hospitalisation are well recognised and appropriately targeted with mechanical and pharmacological thromboprophylaxis (TP) strategies backed by Level I evidence. There is evidence that TP after hospital discharge following surgery (extended TP) is also beneficial. Recently, subgroups such as those with metastases receiving palliative chemotherapy have been shown to be at equally high risk, and also benefit from primary TP. Both chemotherapy and central venous access devices (CVAD) have been shown to be independently associated with development of TE. In addition, early studies examining neoadjuvant radiotherapy (nRT) in rectal cancer reported higher rates of TE however this finding was not repeated in subsequent studies using modern radiotherapy techniques. Extrapolating these findings raises the question of TE risk and the potential benefit of TP during neoadjuvant radiotherapy (nRT) or chemoradiotherapy (nCRT) for rectal cancer. This thesis explores the current evidence and contemporary guidelines concerning TE risk during nCRT, demonstrating considerable uncertainty and a lack of robust data. Randomised trials examining nCRT in rectal cancer are systematically reviewed to determine rates of TE, demonstrating a failure to capture TE events due to inadequate complication reporting frameworks. Existing attitudes and prescribing practices of specialist rectal cancer surgeons are surveyed, as well as barriers to TP prescribing. Issues of equipoise, ownership and logistical problems with outpatient TP prescribing were identified. The historical TE rate and epidemiology over a prolonged follow-up period in rectal cancer patients, as well as the relationship to nCRT is examined at both Peter MacCallum Cancer Centre in Australia and the Cleveland Clinic in the United States. These studies demonstrated that most TE events occurred in patients with metastatic disease receiving ambulatory palliative chemotherapy, and that the overall rate of TE in patients treated with nCRT was not elevated over those patients who had surgery alone. Finally, a prospective study of thrombogenic biomarkers in patients with rectal cancer was undertaken demonstrating significant coagulation abnormalities in colorectal cancer patients at baseline, but no major alteration during neoadjuvant chemoradiation. Marked and prolonged procoagluant abnormalities were demonstrated in the post-operative phase. Thus the current literature, attitudes of treating clinicians, historical rates, as well as candidate biomarkers for prospective TE risk were examined in nCRT for rectal cancer for the first time, identifying future potential research questions aimed at reducing this common complication of cancer care.
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ItemMicropapillary pattern in lung adenocarcinoma: the molecular and clinicopathological characterization of an emerging cancer phenotype( 2015)Adenocarcinoma of the lung is a tumour of mixed morphological appearance under light microscopy with haematoxylin and eosin staining. Last decade, the recognition of a micropapillary pattern by academic pathologists led to its introduction as a subtype in the multidisciplinary pathological classification of a combined taskforce from the International Society for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society. However, its inclusion was controversial, and supported only by a handful of peer-reviewed publications. Little work has been done to further define this pattern beyond its original morphologic description in 2002. Using a top-down approach combining genomic, transcriptomic, immuno-histochemical and clinico-pathological features of a bank of clinically annotated tumours, I sought to define the phenotype and genotype of the micropapillary pattern in lung adenocarcinoma. Additionally I tested the feasibility of next generation RNA sequencing of formalin fixed paraffin embedded (FFPE) tumour tissue. The architecture of micropapillary pattern was confirmed as several variations on the original description of papillary structures lacking fibrovascular cores or small rosettes of cells floating in alveolar spaces, glandular and lymphovascular spaces. The cytology of cells in the micropapillary pattern was one of high-grade nuclear atypia. Using immunohistochemical techniques all micropapillary tumours studied stained for the transcription factor TTF-1. I demonstrated circumferential cytoplasmic staining with MUC1 (a trans-membrane glycoprotein) in micropapillary pattern, as opposed to luminal only or poor staining for other subtypes and variants of adenocarcinoma. The combination of these two stains allowed confirmation of the presence of “spread through alveolar spaces” as another form of micropapillary pattern where small clumps of cells are identified in alveolar spaces of normal lung well away from the main tumour. Intratumoral heterogeneity has been increasingly recognized as a phenomenon in tumours that may be responsible for variable response to therapies and development of resistance. I have demonstrated that individual subtypes within tumours, and especially micropapillary pattern, may harbour mutations not present in the remaining tumour. Additionally, micropapillary tumours had a higher incidence of EGFR and BRAF mutation than adenocarcinomas generally. The differential gene expression profiles of separately sampled subtypes of lung adenocarcinoma were determined from RNA extracted from FFPE tumours using the Nanostring platform. Further evidence of both intratumoral heterogeneity and of a discrete micropapillary phenotype was elicited by the fact that the gene expression signatures of micropapillary pattern generally clustered closer with micropapillary sampled from different adenocarcinomas than they did with other patterns within the same adenocarcinoma. The clinical phenotype of micropapillary predominant adenocarcinoma (MPA) was investigated based on classification by comprehensive histologic subtyping. This involved a quantitative estimate of all subtype patterns in a tumour present in greater than 5% of the tumour volume. Tumours were classified according to the predominant pattern. MPA was found to have the highest rate of lymph node metastasis relative to its presence in primary tumours, and to share the invasive and metastatic potential of solid with mucin predominant adenocarcinoma. These findings give weight to predominant subtype pattern being useful as a form of grading in lung adenocarcinoma, with MPA and solid predominant tumours graded as high grade. Micropapillary pattern of adenocarcinoma appears to be a discrete phenotype with particular genomic aberrations and up-regulation of embryonic stem cell and fibroblast growth factor pathways. Its clinical behaviour is typified by high rate of nodal metastases, spread through alveolar spaces and early relapse and death after adequate surgery.
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ItemOsteochondral repair using structured biological scaffold and stem cell technologies( 2015)Introduction: Articular cartilage damage can result in pain and loss of function for many patients. The traditional management of moderate to severe defects has been difficult due to the lack of intrinsic capacity for cartilage to regenerate. Current methods of cartilage repair include microfracture, osteochondral grafting and autologous chondrocyte implantation. Whilst some individual studies comparing these techniques have shown improvements in long-term clinical outcomes in some patient groups compared to microfracture, major randomised control trials have failed to show consistent long-term differences in clinical outcomes between microfracture, osteochondral grafting, and autologous chondrocyte implantation. Hence, there is a clinical need to explore novel methods of cartilage repair and regeneration using biological techniques such as tissue engineering, stem cells, biomaterials, and growth/differentiation factors to improve cartilage regeneration. The aim of this project was to develop a technique using human infrapatellar fat pad adipose stem cells (IPFP-ASCs) in 3D cultures for chondrogenesis; this required extensive characterisation of the cartilage formed in the 3D cultures/scaffolds (3D pellet culture, chitosan and acellular dermal matrix) for in vitro chondrogenesis. In vivo testing and characterisation of osteochondral defect repair was achieved using a small animal rabbit model for preliminary testing of the ADM-engineered structures. This preliminary testing in the small animal model may then lead to pre-clinical trials in larger animals and human pilot studies in the future. Materials and methods: In vitro IPFPs were harvested from total knee replacements and digested to release adipose stem cells (IPFP-ASCs) which were expanded in vitro. Pellet cultures were developed using TGF-β3 and BMP-6 for chondrogenesis. IPFP-ASCs were seeded onto 3D printed chitosan scaffolds and acellular dermal matrix (ADM) material under the same chondrogenic conditions as the pellet cultures. Four-week cultures were analysed using histology, immunohistochemistry, and gene expression analysis using qPCR. In vivo Osteochondral defects were drilled into distal femoral condyles of adult New Zealand White rabbits. The defects were repaired using either (1) ADM alone (2) autologous IPFP-ASC (3) ADM with autologous IPFP-ASC) or (4) left empty as control. The animals were euthanised at 12 weeks. Repairs were analysed using histology and immunohistochemistry for collagen Type II and Type I. The modified O’Driscoll score was for histological scoring. Further image analysis was conducted to assess quality and quantity of repair. Results: IPFP-ASCs were capable of undergoing chondrogenesis in vitro using pellet cultures and when cultured directly on 3D chitosan and ADM scaffolds using the growth factor combination of TGF-β3 and BMP-6. The method of chondrogenesis was robust and was replicated across both human and rabbit IPFP-ASCs. A one-step single site surgical process was developed for the in vivo modelling of osteochondral defect repair and autologous IPFP-ASC implantation. In rabbits, the rabbit ADM only group achieved the highest ratio of collagen Type II to Type I (77.3%) on image analysis using area measures based from protein expression by immunohistochemistry. This indicated a higher quality of cartilage repair resembling hyaline or hyaline-like cartilage (p<0.05). Conclusion: IPFP-ASCs exhibited robust chondrogenesis under in vitro conditions used in this study; the combination of TGF-β3 and BMP-6 for generation of hyaline cartilage has demonstrated the potential for improving cartilage repair in vitro. ADM, as a support matrix, promoted ASC ingrowth in vitro and proved to be an excellent substrate to promote formation of hyaline-like cartilage in vitro. In the small animal in vivo experiments, it was clear that ADM exhibited positive outcomes when used as a substrate for osteochondral defect repair. These experiments need to be performed in a larger animal model to consolidate these findings prior to consideration of translational to pre-clinical studies.
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ItemEpithelial-to-Mesenchymal Transition (EMT) in human breast cancer: investigating microRNAs in breast cancer EMT( 2015)Breast cancer is the most common malignancy among women worldwide, with mortality primarily associated with metastasis. In recent years, microRNAs (miRNAs) have emerged as a new class of master regulatory molecules with the potential to influence carcinoma progression. They are a class of small RNA molecules that regulate gene transcript stability and processing by binding to discreet motifs in the 3’ and 5’ untranslated regions of mRNAs. They regulate important mechanisms in development, including epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), which have also been associated with cancer metastasis. miRNA profiling of control and EMT-induced PMC42-ET, PMC42-LA and MDA-MB-468 cultured human cell lines using microarray and Next Generation Sequencing (NGS) was undertaken. Several miRNAs were reproducibly up- or downregulated between the untreated cells, and in response to epidermal growth factor. Variations in miRNA expression were also assessed bioinformatically using publically available data from >50 human breast cancer cell lines. Whilst a number of these have already been implicated in cancer, other novel miRNAs consistently associated with EMT/MET were also identified. The expression levels of >20 miRNAs were experimentally validated and stable miRNA manipulations (overexpression and knockdown) in cell lines with low endogenous expression were achieved by lentiviral transduction. Recent evidence has demonstrated a crucial role for the miR-200 family in carcinoma progression, tumourigenesis and metastasis in various cancers including breast cancer. miR-200 regulation of EMT and MET was identified in our microarray and NGS datasets and was characterised in the two EMT models commonly used in our laboratory – the PMC42 and MDA-MB-468 human breast cancer cell lines. In vitro functional changes upon miR-200 manipulation showed a strong correlation between high miR-200 levels and the epithelial phenotype. In vivo changes mirrored the in vitro changes, where miR-200c knockdown resulted in reduced primary tumour growth and increased axillary lymph node metastasis in the MDA-MB-468 xenograft model. Transcriptionally, it was observed that the MDA-MB-468 miR-200c knockdown tumours showed upregulation of epithelial genes (such as E-cadherin, Grhl2, EpCAM) and a corresponding downregulation of mesenchymal genes (such as BNIP3, FN1), suggesting that the MDA-MB-468 miR-200c knockdown cells that were able to survive and eventually form a tumour had to transcriptionally activate epithelial-associated genes to achieve tumour development. Despite this, circulating tumour cells in the peripheral blood of MDA-MB-468 miR-200c knockdown tumour-bearing mice showed indications of a hybrid state, trending towards a more mesenchymal profile. Apart from the miR-200 family, eight other miRNAs—namely miR-744, miR-153, miR-708, miR-483, miR-100, miR-34b/c, miR-146a and miR-29a, were short-listed as candidate EMT-associated miRNAs identified from microarray, NGS and bioinformatic analyses. Collectively our findings suggest that no one particular miRNA alone was capable of functionally altering MDA-MB-468 breast cancer cells. Interestingly, when the cells were driven to a more mesenchymal state, there was a trend towards increased lymph node metastasis, suggesting that a mesenchymal profile is advantageous in that process. The overall findings of this thesis suggest that miRNAs do play a key role in the area of breast cancer EMT and each step in the metastasis cascade can be regulated by different miRNAs. A cooperative network of miRNA changes results in downstream changes in tumourigenesis and metastasis. These studies highlight the need to further investigate context-dependent miRNA manipulations and validate miRNAs that play critical regulatory roles to properly understand the complex role of various important miRNAs in breast cancer EMT.
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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.