Surgery (St Vincent's) - Theses
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ItemNovel mitochondrial Drp1 inhibitors for cardioprotection( 2023)Mitochondria are dynamic organelles, constantly undergoing fusion and fission in a balanced manner to maintain cellular health. In the setting of myocardial ischaemia-reperfusion injury, mitochondrial morphology shifts towards excessive fission, which is associated with cardiomyocyte death and heart dysfunction. Inhibiting the mitochondrial fission protein dynamin-related protein 1 (Drp1) has been shown to reduce excessive mitochondrial fission and attenuate the pathological consequences of myocardial ischaemia-reperfusion injury. However, the most widely used inhibitor, Mdivi-1, is an unreliable inhibitor of Drp1 because of its off-target effects and inconsistent cytoprotection in different cell types, including mammalian cells. Mdivi-1 was originally developed to inhibit the GTPase enzymatic activity of Dnm1, a yeast homologue of human Drp1 protein, which has less than 50% similarity compared to human Drp1. These lines of evidence indicate that Mdivi-1 may not be a specific inhibitor of human Drp1. The overall aim of this thesis is to identify potential inhibitors of Drp1 that directly bind to, and inhibit the GTPase activity of human Drp1, and impart protection against in vitro and in vivo models of acute myocardial ischaemia-reperfusion injury. In Chapter 3, I investigated the interaction between Mdivi-1, yeast Dnm1 and human Drp1 using molecular modelling. Molecular docking analysis predicted that Mdivi-1 is docked more consistently in an open binding site conformation of both species with greater number of molecular interactions between the compound and yeast Dnm1 compared to human Drp1. Biological analysis of Mdivi-1 to human Drp1 was inconclusive due to differing results in direct binding assays, GTPase activity assay and mitochondrial morphology assays in Drp1 wildtype and knockout mouse embryonic fibroblasts. These results are likely confounded by the formation of Mdivi-1 aggregates at concentrations above 18.5 uM. These findings suggest that studies employing Mdivi-1 as an inhibitor of Drp1 warrant cautious interpretation as its effect may not be entirely Drp1-specific. In Chapter 4, further study was then conducted to identify a novel potential inhibitor of human Drp1. The drug discovery campaign for this project had already begun prior to my PhD study and three hit compounds, DRP1i1, DRP1i2 and DRP1i3 were previously identified. The three hit compounds represent three compound classes with distinct scaffolds, namely the diazabicyclic scaffold, tryptophan-like scaffold and the diazaspirocyclic scaffold. Direct binding assays, GTPase activity assays and mitochondrial morphology assays using Drp1 wildtype and knockout mouse embryonic fibroblasts indicate that DRP1i1, DRP1i2 and DRP3 directly bind to human Drp1, can inhibit its GTPase activity and supress Drp1-mediated mitochondrial fission. The most potent hit compound, DRP1i1 (KD value 3.23 uM), was selected for further investigation in in vitro and in vivo models of acute ischaemia-reperfusion injury in Chapter 5. In Chapter 5, DRP1i1 reduced cell death of HL1 cells and human cardiomyocytes derived from induced pluripotent stem cells subjected to hydrogen peroxide-induced oxidative stress and simulated ischaemia-reperfusion injury. In general, this protection was accompanied by reduced mitochondrial fragmentation, decreased mitochondrial superoxide production and improved mitochondrial membrane potential. The protective effect of DRP1i1 was also demonstrated in an in vivo mouse model of acute myocardial ischaemia-reperfusion injury, where I observed a reduction of infarct size accompanied by reduced phosphorylation of Drp1 at Ser616 and reduced circularity of myocardial interfibrillar mitochondria. Collectively, these results suggest that direct inhibition of the Drp1 protein with DRP1i1 possess a cytoprotective effect in in vitro and in vivo models of myocardial ischaemia-reperfusion injury. Due to the moderate affinity of the three hit compounds (within micromolar range; 3.23 uM for DRP1i1, 352 uM for DRP1i2 and 215 uM for DRP1i3), our lab had previously searched for structural analogues of each compound class in a two-dimensional analogue search based on the Tanimoto similarity index of 0.8. A total of 26 structural analogues of DRP1i1, 7 of DRP1i2 and 30 of DRP1i3 were identified. 10 additional analogues of DRP1i2 were also designed by our collaborator, giving us a total of 17 structural analogues for DRP1i2. In Chapter 6, I assessed these analogues for direct binding to human Drp1 and conducted molecular docking studies against human Drp1 to elucidate their structure activity relationship. Molecular docking analysis showed that DRP1i2 and its active analogues displayed the most consistently docked binding mode to the open conformation of human Drp1, whereas analogues of DRP1i1 and DRP1i3 did not show a clear consistency in binding mode. Regardless, hydrogen bond interactions between active compounds and amino acids Lys38 and Ser39 could be important for compound activity in all compound class and the effect of stereochemistry on binding affinity to human Drp1 protein was clearly demonstrated. Among all compound classes, only structural analogues of DRP1i2 and DRP1i3 that could potentially be more potent than their parent compounds. Collectively, the information on the structure activity relationship of these structural analogues will provide the essential fundamental knowledge to design better and more potent inhibitors of human Drp1 in future studies.
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ItemNo Preview AvailableCharacterisation of sensory corpuscles, vasculature and collagen in the injured scapho-lunate ligament( 2022)Background: The scapho-lunate ligament (SLL) is a ligament spanning between the scaphoid and lunate bones of the carpus. It has a well-established role in ligamentous restraint of intercarpal motion and maintenance of carpal alignment under axial load. The SLL also has a role as a sensory organ. Sensory mechanoreceptors are located within the ligament. These receptors are responsible for the afferent input to the reflex arc of extrinsic forearm muscular contraction that provides additional dynamic stability to the wrist joint. The SLL is the most frequently injured ligament within the wrist[1]. Complete disruption of the SLL causes carpal instability and malalignment[2], which eventually leads to osteoarthritis of the wrist. When the ligament is torn, surgical treatment options include reconstruction of the ligament with procedures that usually use tendon grafts or transfers that act as a replacement for critical portions of the damaged ligament complex. These techniques may be able to replicate the static restraint of the SLL, however, they fall short of producing an innervated construct that is capable of co-ordinating neuromuscular reflexes. In search of improved reconstructive surgical options, the ligamentous stump of the damaged SLL has been postulated to be a source of native cells that may drive the process of ligamentisation, the process of transformation of the reconstructed ligament created from alternative tissue such as tendon or synthetic construct, to the histological and functional phenotype of a true ligament. This phenomenon has been investigated in knee ligament reconstruction, with anterior cruciate ligament (ACL) remnant preservation being a popular technique used in ACL reconstruction. Methods: This study aims to investigate the structural and cellular makeup of injured SLLs and compare them to the known structure of normal SLLs to estimate the regenerative potential of the residual SLL stump and its value in developing innervated constructs. Injured SLLs were collected from voluntary human subjects at the time of SLL reconstruction or limited wrist fusion, where the ligament remnants would otherwise be discarded. These specimens were formalin fixed and paraffin embedded for histological analysis and immunostaining was performed to identify the vascular and neural structures of the SLL as well as to determine its collagen constitution. Results: Fifteen ligaments were harvested from subjects at surgery after SLL injury ranging from 39 days to 20 years from time of injury. Eleven ligaments were harvested less than one year after injury and four ligaments were harvested two years or more after injury. A total of 66 mechanoreceptors were identified, 50 in the 11 specimens harvested less than one year following injury. Nine of these 11 specimens within one year contained mechanoreceptors, 54% of which resided in the volar subunit, 20% in the dorsal subunit and 26% in the proximal subunit. Two of the four specimens harvested two years or later from injury contained mechanoreceptors, all of which were located in the dorsal subunit. Blood vessels were found in 13 of the 15 samples. Mean vessel density for all specimens was 1.3%, with the highest average density being 1.8% for the volar subunit. The vessel density in the volar subunit decreased with time after injury, with vessel density of 2.5% seen in specimens less than three months post injury, and vessel density of 0.03% in those specimens that were more than three months post injury. The mean collagen I density was 45.6% of the ligament area for all specimens, with the highest average in the dorsal subunit (53% of the ligament area). Collagen I density did not vary significantly within the first two years of injury. Collagen III density varied significantly with time since injury, with average density of 47% of ligament area in specimens less than three months after injury and and 31% in those specimens that were harvested greater than three months after injury. This decline in Collagen III density after three months was most prominent in the volar subunit. Collagen I:III ratio reflected the pattern of change seen in the collagen III profile. Conclusion: Mechanoreceptors were reliably located in the SLL, particularly in the volar subunit of specimens harvested less than one year after injury. Vessel density in the volar subunits was reliably associated with the mechanoreceptor distribution, as was collagen III distribution in the volar subunit in the initial three months post injury. Scapholunate ligaments demonstrate favourable histological findings that warrant further research into remnant inclusion techniques in earlier injuries, particularly incorporating the volar subunit.
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ItemThe role of digital technology and gamification to improve health literacy in patients undergoing arthroplasty( 2022)Arthroplasty is a high-volume but costly treatment option for end-stage osteoarthritis. This PhD used a mixed methods approach to explore new strategies to better select and prepare surgical candidates for their surgical journey. A narrative review conveyed how health literacy can improve understanding, rationalise expectations and reduce dissatisfaction in arthroplasty through the medium of digital technology and gamification (the use of gaming elements in a non-gaming context). A quantitative analysis of online arthroplasty information quality demonstrated a marked shortage of reliable resources for patients. A scoping review revealed how existing digital interventions can have a positive impact on related aspects of health literacy, such as knowledge and self-management, despite no structured approach or theoretical framework around these designs. An observational study determined the health literacy profile of a patient cohort undergoing arthroplasty, in whom were lacking the abilities to actively manage their health, and find and appraise health information. It also found that participants who utilised the internet often had higher health literacy, and those able to actively self-manage were three times as likely to progress to surgery. Qualitative research with people before and after their orthopaedic surgery consultation, found those with higher health literacy (including the ability to actively self-manage) had already made the decision to undergo surgery and reflected better surgical candidacy to the surgeon. Finally, interviews with patients about their attitudes, usage and opinions towards a digital tool found that the most effective digital education tool included practical clinical, logistical and lifestyle information, including checklists and timelines, combined with nuanced gamified mechanics, such as points, badges and self-tracking data. The findings from these six studies were synthesized into a concept design for a digital, gamified education tool. It is a data-driven design which ultimately aims to improve health literacy in the context of arthroplasty, empowering patients to obtain evidence-based knowledge, and seek and receive the right treatment at the right time.
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ItemAssessing the suitability of Australian general practice electronic health record data for clinical prediction model development: A case study in Osteoarthritis( 2022)Australian general practice electronic health records contain a wealth of patient information. However, the suitability of these data for developing clinical prediction tools is unclear. This research focussed on determining the suitability of these data for prediction model development using osteoarthritis as a case-study. A comprehensive data quality assessment was conducted and a prediction model for total knee replacement surgery developed. The thesis demonstrated that suitability of Australian general practice electronic health record data for prediction model development should be assessed on a case-by-case basis. A decision aid was developed to assist researchers in determining whether their electronic health record data are fit for prediction model development.
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ItemBio-engineering vascularised liver organoids( 2022)Liver organoids are bioengineered constructs that recapitulate native liver tissue, and are used to study liver development, test drugs, and as replacement tissue that can be transplanted to treat liver disease. This thesis focusses on the specific application of liver organoids for use as a cell-based treatment for liver disease via organoid transplantation. It places a particular emphasis on the addition of vascularisation to liver organoids to enhance structure and function, and improve engraftment during in vivo transplantation. Initially, the effect of the addition of endothelial cells to create vascularised liver organoids was assessed using mouse cells. The addition of mouse liver sinusoidal endothelial cells (LSECs) to mouse liver progenitor cells (LPCs) resulted in a striking change in organoid morphology, with the development of hepatobiliary ductular structures and clusters of polygonal hepatocyte-like cells which did not appear when LPCs were cultured alone as organoids. Furthermore, in vitro hepatobiliary gene expression, hepatic synthetic functions (albumin and apolipoprotein E production) and organoid viability was significantly increased by the addition of LSECs. Upon transplantation into vascularised chambers established in Fah-/- Rag2-/- Il2rg-/- (FRG knockout) mice, LPC only organoids had almost zero survival at 2 weeks, whereas LPC/LSEC organoids developed robust hepatobiliary ductular structures with a 115-fold increase in HNF4a+ cells and 42-fold increase in Sox9+ cells. To translate the mouse findings into a humanised platform, human LPCs and LSECs and their human induced pluripotent stem cell (hiPSC)-derived counterparts were characterised. The hepatic differentiation of human primary adult LPCs and hiPSC derived LPCs into hepatocyte-like cells was confirmed based on cell morphology, marker expression, and function (albumin production), and transcriptomic profiling using bulk and single cell RNA sequencing. Concurrently, human primary adult LSECs were compared to hiPSC-derived endothelial cells(iECs). Although in vitro iECs had a generic endothelial phenotype very different to LSECs, when iECs were transplanted into mouse liver they underwent tissue specification to approximate LSECs, highlighting the importance of the liver microenvironment in this process. Subsequently, three types of vascularised human liver organoids were explored using primary human and hiPSC-derived cells. First, primary LPCs, LSECs and adipose-derived mesenchymal stem cells were combined in a human liver-derived extracellular matrix (ECM) hydrogel and seeded into bioabsorbable porous polyurethane scaffolds. Second, iECs were aggregated with hiPSC-derived hepatocyte (iHep) organoids to coat the surface of the iHep organoids. Third, single cell-type organoids were integrated to form a combination organoid created from hepatocyte, cholangiocyte, and vascular organoids. Of the three models, combining organoids of different cell types to create a combination organoid was deemed the best approach to derive complex liver organoids containing well-organised tissue structures such as polarised hepatocytes with bile canaliculi, bile ducts, and blood vessel networks. An overarching theme is that vascularisation is pivotal in the development of transplantable liver organoids. Adding endothelial cells promotes hepatobiliary differentiation, and pre-formed vasculature can significantly enhance the survival of transplanted liver organoids by hastening connection to the host’s blood supply. However, this is not easy to achieve and remains a challenge in the liver organoid field, and the production of well-vascularised liver organoids with sustained development of blood vessels over time in culture remains elusive. Nevertheless, the limitations and challenges identified in this thesis point towards future directions in addressing the issue of vascularisation. For clinical translation of liver organoid transplantation, hiPSC-derived cells are a more reliable source of personalised cells, and the ECM additive should be bio-synthetic and chemically-defined, rather than Matrigel. Ultimately, the results in this thesis support the exciting prospect of stem-cell derived liver organoids being used as a regenerative treatment for liver disease.
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Item‘Every bone in my body aches’: A culturally secure exploration of osteoarthritis among Aboriginal and Torres Strait Islander People( 2022)Background As a leading cause of pain and disability worldwide, osteoarthritis is associated with considerable personal, economic and societal toll. Osteoarthritis commonly coexists with chronic diseases such as heart disease, diabetes and mental health challenges and can exacerbate the morbidity and mortality associated with these conditions. The health and social disparities experienced by Aboriginal and Torres Strait Islander people in Australia are well documented and include a greater burden of chronic disease, poorer self-assessed health status and lower life expectancy. Osteoarthritis and the burden of illness among Aboriginal and Torres Strait Islander people represent two substantial public health challenges today, however there is a dearth of research which bring the two together. The limited research available suggests that Aboriginal and Torres Strait Islander people experience a higher prevalence and greater burden of osteoarthritis, yet access health care for osteoarthritis at substantially lower rates than non-Aboriginal people. With no culturally adapted models of osteoarthritis care for Aboriginal and Torres Strait Islander people, osteoarthritis is an unmet health need. This thesis responds to these gaps by exploring osteoarthritis among Aboriginal and Torres Strait Islander people and laying the foundations of culturally secure osteoarthritis research for Aboriginal and Torres Strait Islander people. Cultural security in research occurs when the research is conducted in a way that will not compromise the cultural rights, values, beliefs, knowledge systems and expectations of Aboriginal and Torres Strait Islander people. Objectives To explore osteoarthritis among Aboriginal and Torres Strait Islander people and lay the foundations of culturally secure osteoarthritis research for Aboriginal and Torres Strait Islander people, the aims of this PhD were to; 1) Establish an Aboriginal and Torres Strait Islander community reference group to inform, guide and enable community engagement throughout the research; 2) Examine the access, utilisation and surgical outcomes among Aboriginal and Torres Strait Islander people with hip and knee osteoarthritis undergoing total joint replacement surgery and; 3) Inform and guide the development of culturally secure osteoarthritis care for Aboriginal and Torres Strait Islander people based on their lived experience. Methods To achieve these aims, a mixed-methods approach, situated within methodological pluralism and a pragmatic epistemology was employed. Predominantly qualitative methods were chosen to prioritise the stories and cultural context of Aboriginal and Torres Strait Islander participants. This PhD was also conducted within a culturally secure research framework, which prioritised relationships, community engagement and Aboriginal and Torres Strait Islander cultural values and voices throughout. Four phases of research were conducted: 1. In Phase 1, a review of the literature describing osteoarthritis and health care access, including access to total joint replacement among Aboriginal and Torres Strait Islander people was performed. 2. In Phase 2, a qualitative key informant study was conducted. Research yarning interviews with Aboriginal health stakeholders and Aboriginal and Torres Strait Islander people with lived experience of osteoarthritis were conducted to inform the establishment of a community reference group and terms of reference. 3. In Phase 3, a qualitative dominant mixed-methods study was conducted. First, a retrospective cohort study was conducted to explore the uptake and utilisation of total joint replacement by Aboriginal and Torres Strait Islander people and characteristics of Aboriginal and Torres Strait Islander people undergoing total joint replacement. This was followed by a qualitative exploration of the lived experience of osteoarthritis from the perspective of Aboriginal and Torres Strait Islander people, and; 4. In Phase 4, data collected in Phase 3 were integrated to inform and guide the development of culturally secure osteoarthritis care and to develop the discussion chapter of this thesis. Findings The review of the literature conducted in Phase 1 highlighted a lack of evidence describing osteoarthritis among Aboriginal and Torres Strait Islander people, particularly from a person-centred perspective. Two published manuscripts addressed the paucity of research directed towards this unmet health need. The first publication ‘Tackling the burden of osteoarthritis as a health care opportunity in Indigenous communities – A call to action’ raises the profile of osteoarthritis as an unmet health need for Indigenous communities around the world, placing this public health issue on the research agenda. The second narrative review and call to action titled ‘Addressing surgical inequity for Aboriginal and Torres Strait Islander people in Australia’s universal health care system: A call to action’ highlights that Aboriginal and Torres Strait Islander people experience significant barriers to accessing care along the whole of the patient journey to surgical care by drawing examples from the field of osteoarthritis and total joint replacement. The process of establishing an Aboriginal and Torres Strait Islander community reference group and terms of reference in Phase 2 was described in the manuscript (under review) ‘Laying the foundations of community engagement in Aboriginal health research: Establishing a community reference group and terms of reference in a novel research field’. Trust and relationships were considered the most important factors in successfully recruiting ‘the right people’ to an Aboriginal and Torres Strait Islander community reference group. Key informants recommended that representatives of a broad spectrum of expertise should have a seat at the table, including both health service providers and Aboriginal and Torres Strait Islander people with lived experience of osteoarthritis. Community engagement, research steering, knowledge dissemination and advocacy were identified as essential functions of a community reference group. Emphasis was placed on the importance of self-determination, ownership and the decision-making power of the group to negotiate their own terms and conditions. The findings from this study informed the establishment of an Aboriginal and Torres Strait Islander community reference group (comprised of six members) and terms of reference who provided significant input in all subsequent studies in this PhD. The uptake and characteristics of Aboriginal and Torres Strait Islander people undergoing total joint replacement explored in Phase 3 was presented in the manuscript (under review) ‘Total joint replacement may be a valuable treatment for Aboriginal and Torres Strait Islander people with osteoarthritis, but uptake is low’. This manuscript describes the findings from a registry-based retrospective cohort study which compared the sociodemographic, clinical characteristics and surgical outcomes of all Aboriginal and Torres Strait Islander patients and non-Aboriginal patients who underwent primary hip and knee replacement at St Vincent’s Hospital Melbourne between 1996-2019. This exploratory study identified that in the 23-year study period, 49 (0.5%) of procedures were conducted in patients who identified as Aboriginal, Torres Strait Islander or Aboriginal and Torres Strait Islander. St Vincent’s Hospital Melbourne is the largest metropolitan provider of care to Victoria’s Aboriginal community, with over 5000 occasions of care to Aboriginal adults annually. Based on hospital data from the Decision Support Unit, approximately 2.2% of acute hospital admissions at SVHM are patients who identify as Aboriginal, Aboriginal and Torres Strait Islander or Torres Strait Islander. Aboriginal people make up approximately 1.6% of the population in Victoria, excluding metropolitan Melbourne, and 0.5% of the population of Melbourne. The study also found that although Aboriginal and Torres Strait Islander people undergoing total joint replacement were younger, more likely to be overweight and more likely to have a diagnosis of diabetes or multiple chronic diseases, they experienced comparable post-operative quality of life improvements 12-months post-surgery to non-Aboriginal people. The lived experience of osteoarthritis from the perspective of Aboriginal and Torres Strait Islander people, also investigated in Phase 3, was described in the manuscript (accepted for publication) ‘Understanding the impact and tackling the burden of osteoarthritis for Aboriginal and Torres Strait Islander people’. This study identified that osteoarthritis is a multidimensional issue for Aboriginal and Torres Strait Islander people which permeates all aspects of life. Many participants experienced significant psychosocial impacts of osteoarthritis which often occurred within already stressful or complex health or life circumstances. Participants also described multiple negative health care experiences whilst seeking care for their osteoarthritis, which were often characterised by ineffective communication by health care providers. Conclusions As the first in-depth person-centred exploration of osteoarthritis among Aboriginal people and Torres Strait Islander people, this research highlighted the impact of osteoarthritis as a significant health concern which permeates all aspects of life for those affected and validates the importance of addressing the impacts of osteoarthritis within a multidimensional framework. Integrated, multidisciplinary care which is evidence based, culturally secure, individualised to patient need and accessible is required to address the burden of osteoarthritis among Aboriginal and Torres Strait Islander people. By addressing the burden of osteoarthritis among Aboriginal and Torres Strait Islander people, there is an opportunity to improve the health and wellbeing of Aboriginal and Torres Strait Islander communities.
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ItemTackling regulation “In situ”: A model for approaching regulation during a 3D bioprinting pre-clinical research translation program( 2021)3D bioprinting uses the techniques of additive manufacturing, but includes living cells, with the goal of creating living 3D tissues for modelling disease or for patient implantation. This ongoing technological revolution presents a major challenge for regulators as the current regulatory frameworks are designed for mass manufactured, standardised devices, and as such are not suited to 3D bioprinted, individual-specific devices, often using the patient’s own cells. The aims of the project were to identify and highlight the challenges of regulating 3D bioprinting technologies, and to develop an approach and the relevant tools that researchers could use for integrating regulatory considerations into the development pipeline for a 3D bioprinting device during a research translation program. By using the Axcelda (formerly known as the Biopen) project example and associated publications, this research highlighted several areas of commonly missed opportunity to consider and integrate relevant regulatory requirements into the initial academic study design. These included both general and specific considerations such as the generation of technical and regulatory documentation to better outline the existing product and components, initial critical ingredient identification and safety profiling, risks and regulatory strategy, as well as to incorporate additional specific biocompatibility testing into early pre-clinical studies, among other considerations. Doing so demonstrated the potential of this process to lead to better designed, faster and more efficient studies (from a regulatory perspective), thus increasing the potential for successful translation into a commercial product.
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ItemMultiparametric MRI in Prostate Cancer Diagnosis and Management( 2021)Prostate canceris the most common cancer affecting men in Australia. Detection has previously relied upon clinical examination and prostate specific antigen (PSA) screening. Addition of MRI of the prostate prior to prostate biopsy has increased our ability to accurately diagnose prostate cancer. This thesis examines the implementation of multiparametric MRI of the prostate in Australia and the impact it has had on prostate cancer diagnosis.
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ItemBiofabrication of articular cartilage: Development of an efficient in vivo repair technique using autologous stem cells( 2020)Articular cartilage defects represent a major clinical challenge due to the lack of long-term management options available for young patients who present with a symptomatic and functional burden. Microfracture is the traditional standard treatment of care and has no long-term benefit demonstrated beyond 2 years, with patients reporting symptom relapse and functional compromise. Other techniques used to treat chondral defects include Autologous Chondrocyte Implantation and Matrix-induced Autologous Chondrocyte Implantation, both of which are not superior in comparison to the cheap and easily performed microfracture technique. Cartilage tissue engineering approaches using stem cells and bioscaffolds have become of significant research focus; additionally, the emergence of bioprinting technology has opened up the ability to efficiently and accurately deliver engineered tissue constructs. Biological tissue can be generated by printing cells and scaffolds together in a ‘bioink’ composition rather than using prefabricated scaffold constructs; this approach is coined ‘Biofabrication’, which is a rapidly growing field. Biofabrication approaches show promise in treating chondral defects; however, we are no closer today to a human clinical trial. Several hurdles currently prevent the progression of such research; a significant barrier is the use of long periods of laboratory-based cell culture and expansion. This increased culture duration leads to concerns with the use of animal serum-based media, sterility, senescence, loss of differentiation potential, and tumorigenic transformation. To overcome these issues, human tissue harvest, cell isolation and reimplantation should be performed efficiently, thereby reducing the exposure to the risks mentioned above. Furthermore, by establishing a specific timeframe in which a biofabrication procedure can be achieved, surgical planning and patient preparation can be structured and adequately performed. This thesis aimed to develop an efficient biofabrication procedure for cartilage repair using an autologous cell population, which could produce neocartilage in clinically relevant defects. The chapters in this work present several critical developments concerning the overall aim. First, the most chondrogenic cell source from those tested within the knee joint was identified to be the human Adipose-Derived Stem Cell (hADSC). A rapid 85-minute hADSCs isolation protocol from the Infrapatellar Fat Pad (IFP) was then developed by optimising the time-consuming aspects of the standard IFP-derived hADSCs isolation protocol (>27 hours) and shown to be comparable. Secondly, the minimum chondrogenic requirements of rapidly isolated hADSCs before reimplantation were established. It was determined that 5 days is the earliest time point during cellular expansion in which hADSCs could be driven into chondrogenesis. Therefore, the minimum biofabrication turnaround time is roughly 1-week (5 days and 85 minutes to be precise). Next, 5.0 million hADSCs/mL of a biocompatible hydrogel was shown to be the minimum concentration required to produce in vitro neocartilage. Finally, the maximum defect volume treatable in a 1-week turnaround was shown to be 380 uL (mm3) or 760 uL (mm3) using one or two IFPs respectively, representing clinically significant defect volumes. The next section of this thesis aimed to establish a biofabrication model that could be adapted for surgical use and be implemented in an animal model. In this chapter, a safe, efficient and user-friendly procedure was designed and validated in vitro. First a representative cell source was selected and validated. Next, suitable hydrogel compositions and gelation times were identified, and finally, a safe intraoperative crosslinking set-up was developed. The final element of this work was a proof of concept study, where the newly devised biofabrication approach was performed on a rabbit model to evaluate chondral repair. This procedure was successfully implemented, and the associated degree of cartilage repair was superior compared to the microfracture (clinical standard) and empty control groups. In conclusion, an efficient 1-week biofabrication approach was established for chondral repair, and this approach was shown to treat clinically significant defect volumes. The newly developed procedure has been validated for short term repair in vivo and is superior to the existing standard treatment. The next step is to provide mid-long-term efficacy of therapy in vivo using a large animal model, which if successful, paves the way to human translation. This work presents promise in the future management of chondral defects in young patients with a low-risk strategy that could one day treat/halt the progression to early-onset osteoarthritis.
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ItemColorectal cancer in rural regional Australia( 2017)Colorectal cancer (CRC) is the second commonest cancer in Australia. The survival outcome of colorectal cancer patients within Australia is reported to vary with population density and between health services, with some literature showing poorer prognosis in rural regional and remote patients. Chapter one aims to outline some key issues in CRC such as epidemiology, diagnosis, treatment, surveillance, and outcome whilst chapter two aims to present a systematic review of how geographical disparity influences CRC survival. There are many potential factors that contribute to a poorer prognosis in rural regional CRC patients though the literature is limited, and at times, inconsistent. Thus, there is a need for regular audit, reporting and benchmarking of outcomes in CRC patients against agreed standards. I reviewed the long-term outcomes of CRC at Barwon Health, which serves the South West Victoria, a region with a population of some 500,000. My aim was to determine whether changes introduced to the management of CRC translated into improved survival after surgery. The literature to date has suggested that patients living in rural and regional Australia (grouped together) have worse colorectal cancer survival rates than those living in metropolitan Australia. This thesis was based on a prospectively maintained registry kept over a period of thirteen years from 2002 to 2014, that had accumulated 1079 patients who had undergone surgery at the University Hospital Geelong for CRC (744 colon cancers and 335 rectal cancers). The overall number of operations per year increased over time (p=0.037) but with similar proportions of elective and emergency surgery (p=0.75) and tumour stage (P=0.21). This lack of change in the proportion of elective cases was in spite of the Federal Government introducing a National Bowel Cancer Screening Program in 2006. The proportion of patients with severe comorbidities did increase (p=0.015) over the study period. The median survival after surgery by stage was 123 months, 141 months, 76 months and 17 months for stages I to IV CRC respectively. Overall, there were improvements observed in both peri-operative mortality (POMR) (p=0.028) and long- term survival (p=0.0025) of CRC patients in this major regional centre. I then reviewed the outcome of patients with metastatic disease. The Geelong database included 843 patients who had undergone resection and primary anastomosis for their primary tumour (661 colon cancers, 182 rectal cancers). Metastatic disease was present in 16% (135 patients) and was associated with an increased risk of anastomotic leakage (13% vs. 5%, p=0.003) and a higher peri- operative mortality rate (9.6% vs. 2.8%, p=0.0003). Patients with anastomotic leakage had a reduction in the overall survival (121 months vs. 66 months, p=0.02). The fifth chapter aimed to perform a regional study to identify patients with colorectal cancer at higher risk of developing metastatic disease. There were 503 patients (345 colon and 158 rectal) with non-metastatic (stage I-III) CRC who had resections and were followed up for at least five years. Metastatic progression was, as expected, significantly higher for patients with stage III disease (aHR 4.42 for colon cancer 95% CI 1.74 to 11.23, aHR 3.34 for rectal cancer 95% CI 1.36 to 8.22), and those with lymphovascular invasion (aHR 2.94 95% CI 1.70 to 5.06). Metastatic disease was also more likely to eventuate in those with severe comorbidities (aHR 2.18, 95% CI 0.26 to 0.86), and in colon cancer patients with the lowest socioeconomic status (aHR 2.03 95% CI 1.23 to 3.34). Gender, tumour location and geographical location (rural or regional) was not associated with metastatic progression. Before determining surveillance strategies targeting higher-risk patient groups in regional Victoria, these findings would require confirmation from similar studies in other regions of rural/regional Victoria, such as Bendigo, Albury / Wodonga, Latrobe Valley, or Shepparton.
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