Surgery (St Vincent's) - Theses
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ItemTechniques and technologies in joint replacement surgery: evaluating the value proposition of computer navigation in total knee replacement( 2023-01)This thesis examined the role of computer navigation technologies in total knee replacement. I provide an overview of osteoarthritis and its impact across our healthcare system, the role of total knee replacement in the current management of knee osteoarthritis, and the value that total knee replacement delivers. I then examined the value proposition of computer navigation technologies used in total knee replacement, and the implications of this with respect to current surgical practices in total knee replacement. This was undertaken through a range of perspectives including patient-reported outcomes, complications, and resource utilisation. Finally, I evaluated the cost-effectiveness of computer navigation in total knee replacement surgery through a decision analysis using a Markov-based model informed by my preceding works. This body of work relied largely on the St Vincent’s Melbourne Arthroplasty Outcomes Registry (SMART), an institutional lower limb joint arthroplasty registry, based at St Vincent’s Hospital Melbourne under the stewardship of the University of Melbourne Department of Surgery and the Department of Orthopaedic Surgery at St Vincent’s Hospital Melbourne. I employed a variety of statistical and health economic strategies in performing these investigations and utilised propensity-score methods to ensure that the analyses conducted herein formed a valid and robust contribution to expanding the literature on techniques and technologies in joint replacement surgery.
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ItemOptimising Preoperative Decision-Making in Total Knee Arthroplasty Using a Machine Learning Approach: Development, internal validation, and clinical acceptability evaluation of a clinician-informed machine learning model for the prediction of 30-day readmission following total knee arthroplasty( 2023-06)Background: Total knee arthroplasty is an effective treatment for advanced osteoarthritis of the knee joint, leading to reduced pain, improved function, and better quality of life for affected patients. Following a total knee arthroplasty (TKA) procedure, 30-day readmissions indicate a suboptimal postoperative course which negatively impacts upon the patient’s recovery and poses a significant burden to the healthcare system. Machine learning techniques can be used to predict readmission risk for individual patients and therefore can be implemented in tools to support shared clinical decision-making between patient and orthopaedic surgeon. Objectives: 1. To utilise the experience and expertise of clinicians involved in the care of TKA patients in the identification and appraisal of risk factors for 30-day day readmission. 2. To develop a statistical model to predict 30-day readmission in TKA patients, utilising machine learning techniques and clinical insight for use in shared clinical decision-making. 3. To evaluate the performance of clinicians regularly involved in the care of TKA patients on predicting 30-day readmission following TKA for individual patients then compare the predictive performance of a risk prediction model with that of clinicians. 4. To explore the understanding of TKA patients regarding what AI is and what are its perceived benefits and potential pitfalls in the context of shared clinical decision-making. Methods: Mixed methods approach involving five stages, adapted from literature pertaining to the development and implementation of complex interventions. Stakeholder involvement was utilised throughout the project to engage clinicians, hospital administrative staff, and patients themselves. Patient involvement was embedded throughout the project by means of a research buddy program, and this was detailed in a perspective piece included in the Methods. Stage 1 involved risk factor identification and evaluation, comprising two stages: first, a narrative review, systematic review protocol, and systematic review and meta-analysis on patient-related risk factors for 30-day readmission following TKA; second, a modified Delphi survey and focus group study based on systematic review findings. Stage 2 involved dataset acquisition and description, comprising a cohort profile for the institutional arthroplasty registry and a narrative description of the process of accessing and utilising hospital administrative data. Stage 3 involved a multivariable predictive model development study based utilising machine learning techniques as well as clinical insight gained in Stage 1. Stage 4 involved clinical acceptability evaluation in the form of a computer vs clinician comparison study. Finally, Stage 5 involved clinical acceptability evaluation, capturing the patient perspective in a qualitative semi-structured interview study. Findings: Clinicians provided insight into the complexity of predicting readmission on account of the diverse range of risk factors. Together with machine learning and statistical techniques, this insight was applied to arthroplasty registry and hospital administrative data to develop a predictive model which i) outperformed clinicians’ predictive capabilities and ii) was adequately calibrated to facilitate implementation in the clinical setting. The qualitative study, co-designed with a consumer advocate, found that TKA patients were open to the use of AI in shared clinical decision-making, and these findings were contextualised in prior literature to generate recommendations for future implementation. Conclusions: This thesis demonstrated the development of a bespoke readmission risk prediction model for TKA patients in a process involving broad stakeholder involvement in recognition of the intrinsic value of involving stakeholders in research and development initiatives that impact upon them, and in recognition of the responsibility of researchers to do so. This process primed the model for future implementation to enhance shared clinical decision-making.
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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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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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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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ItemPre-mRNA alternative splicing in the epithelial to mesenchymal transition of breast cancer cells( 2018)Pre-mRNA alternative splicing in the epithelial to mesenchymal transition of breast cancer cells Edwin Widodo, Eva Tomaskovic-Crook, Bryce van Denderen, Erik W. Thompson Summary Alternative pre-messenger RNA splicing is a process that generates multiple variants of a single gene by virtue of the alternative exons that are transcribed. In breast cancer progression and metastasis, alternative splice events (ASE) are regulated during epithelial to mesenchymal transition (EMT). EMT occurs naturally during embryonic development as epithelial-derived cells become transiently mesenchymal and move around the embryo to generate the body plan. EMT status can be determined by expression of specific markers for EMT. E-cadherin is recognized as the archetypical marker for the epithelial phenotype. During carcinoma EMT, E-Cadherin is reduced by transcriptional repression and/or translocation away from the membrane junctions, and the cytokeratin intermediate filament network is reduced or lost while vimentin expression is enhanced. EMT manipulation can be implemented by inducing overexpression of EMT-regulating driver genes, including Twist1 and Snail1 (Mani et al., 2008), and is prominently driven by transforming growth factor beta (TGFbeta). These genes act by transcriptional repression of E-Cadherin. The nuclear factor kappa B (NF-kB) pathway has also been shown to be involved in EMT in MCF10A breast cancer cells. Human breast cancer cell lines are mostly divided into 5 categories based on their characteristics defined in clinical breast cancer datasets. The categories are Luminal A, Luminal B, Basal A, Basal B, and HER2+ types. Hierarchical clustering of high throughput array studies conducted on 34 (Charafe-Jauffret et al., 2006) and 51 (Neve et al., 2006) measuring RNA expression on human breast cancer cell lines grouped those cell lines into Luminal and Basal subgroups. Luminal cells often express estrogen receptor (ER+) and progesterone receptor (PR+) while Basal cell lines lack expression of ER, PR and HER2 (triple negative) and are more resistant to adjuvant chemotherapy. The Luminal group was further divided into Luminal A with low Ki67, a marker of proliferating cells, and Luminal B with high Ki67. The Basal group of cell lines was further divided into 2 groups, Basal A and Basal B (Neve et al., 2006). Most cell lines in the Basal B group have a more invasive phenotype and exhibit a mesenchymal gene signature. We applied a panel of cell lines from those different molecular subgroups: Luminal (MCF7, which has epithelial features), Basal A (MDA-MB-468) and Basal B (MDA-MB-231, which has mesenchymal properties). The EMT features in the PMC42 system include down-regulation of CDH1 and up-regulation of Vimentin for PMC42-ET and PMC42-LA cell lines (Ackland et al., 2001, Ackland et al., 2003). PMC42 system consists of the parental PMC42-ET and the epithelial subtype, PMC42-LA, the PMC42 system provides us with a spectrum of EMT associated changes. The PMC42-LA cell line contains a low number (10-15%) of Vimentin-positive cells, whereas the PMC42-ET cells are 100% Vimentin positive, with commensurate CDH1 differences (Hugo et al., 2007). Further, in response to EGF PMC42-LA cells undergo EMT-like changes (Ackland et al., 2003). ASE in the PMC42 human breast cancer EMT were investigated by (i) comparisons between the more mesenchymal parental PMC42-ET (ET) cells and the more epithelial PMC42-LA (LA) subline, and (ii) in response to epidermal growth factor (EGF), which stimulates EMT-like changes at the mRNA and protein level in both PMC42 variants. We assessed these effects in 2D monolayer culture as well as 3D cultures in Matrigel or Collagen (Vitrogen) and found very similar results in all three culture conditions. ASE are regulated by Epithelial Splice Regulatory Proteins (ESRP) 1 and 2. The expression of both, ESRP1 and ESRP2, was found to be suppressed in Basal B but not in Luminal or Basal A cell lines (Warzecha et al., 2009a). This suggests that their suppression could be involved in EMT-related events. ESRP1 and 2 mRNA levels were constitutively lower in the mesenchymal ET cells compared to LA, but showed little EGF regulation. ESRP1 mRNA levels in epithelial MCF-7 cells were similar to LA, while mesenchymal MDA-MB-231 cells were similar to ET. For ESRP2, MCF-7 levels were higher than LA. Mammalian Ena Homolog (MENA) levels in both PMC42 variants resembled MCF-7 cells, however both variants predominantly expressed the mesenchymal-associated form, as was the case with Cluster of Differentiation 44 (CD44), Ral GEF with PH Domain and SH3 Binding Motif 2 (RALGPS2) and Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1 (MAGI1). Thus, EMT-associated-ASE revealed predominantly mesenchymal-specific splicing patterns despite the ESRP1 differential, perhaps due to a lack of ESRP2. The results confirm an ESRP1/2-related mesenchymal shift from PMC42-LA to ET cells. In general, we also confirmed a high level of ESRP1 and Fibroblast growth factor receptor 2 – exon IIIb (FGFR2 IIIb) in Luminal and Basal A cells, and reduced level of ESRP1 and higher Fibroblast growth factor receptor 2 – exon IIIc (FGFR2 IIIc) in Basal B cells. The shift from FGFR2-IIIb to FGFR2-IIIc in EMT showed alternatively spliced variants from the same gene, FGFR2. Expression of FGFR2-IIIb measured by splice-specific RT-PCR followed the same pattern as ESRP1, while both PMC42 variants were higher than MCF-7 cells for mesenchymal-associated FGFR2-IIIc. Zinc finger E-box-binding homeobox 1 (ZEB1) mRNA levels, a transcription factor that binds E-box motifs in promoters, were reduced by expression of a short hairpin RNA (shZEB1) in PMC42-ET cells. Lack of ZEB1 resulted in a significant reduction (p=0.0018) of ESRP1, but not ESRP2, consistent with the E-box in the ESRP1 proximal promoter. Although FGFR2 IIIb was upregulated after ZEB1 silencing (p=0.058), FGFR2 IIIc, which was supposed to be alternatively spliced, remained at the same level after ZEB1 silencing (p=0.6263). This suggests a direct role of ZEB1 in ESRP1 expression. Total Enabled Homolog (ENAH) was not reduced significantly after ZEB1 knockdown, (p=0.366). In summary, ZEB1-knockdown in PMC42-ET cells caused enhanced levels of ESRP1 and FGFR2 IIIb expression. Partek Genomic Suite analysis of the Affymetrix data indicated a selective upregulation of a 3’-truncated isoform of Laminin subunit alpha 3 (LAMA3 variant 2 or LAMA3v2) by EGF. qRT-PCR analysis revealed that both the long variant (LAMA3v1) and shorter variant (LAMA3v2) showed enhanced levels along the Luminal to Basal B spectrum (as explained in Chapter 1.1.3.), although the Basal B MDA-MB-231 cells appeared to under-express LAMA3v2. LAMA3v2 was particularly highly expressed in the PMC42 variants and was upregulated by EGF in PMC42-LA cells but not in the PMC42-ET cells. LAMA3v1 levels in PMC42-LA and –ET cells both resembled the MDA-MB-231. In the MDA-MB-468 model of EGF-induced EMT, LAMA3v1 was stimulated by EGF treatment (7 days) but not hypoxia (3 days), whereas LAMA3v2 expression was stimulated by either EGF (7 days) or hypoxia (3 days) treatments. Our group has conducted an experiment by xenografting MDA-MB-468 in mice. In MDA-MB-468 xenografted tumours, LAMA3v2 was expressed significantly higher than LAMA3v1. Gene silencing using small interference RNA (siRNA) techniques provide a sight on the short term effects of knocking down gene(s) in cells. In assessing the effectiveness of silencing subunit alpha 3, subunit beta 3 and subunit gamma 2 of Laminin (LAMA3, LAMB3 and LAMC2), we used short interference RNA (siRNA) targeting LAMA3 (siLAMA3), LAMB3 (siLAMB3) and LAMC2 (siLAMC2) and combination of those three siRNAs (siLAMA3B3C2). On the one hand, inhibition of LAM using the siLAMs, as confirmed with inhibition of LAMB3 LAMC2, and laminin v2, inhibited the expression of EMT markers: Vimentin. On the other hand, siLAM increased expression of Zeb1, ENAH and laminin v1. This may suggest that targeting Laminins using siRNA could reduce the EMT properties of PMC42 cell lines. RNA Seq results showed several top genes as potential candidates for EMT in PMC42 breast cancer cells. We applied Multivariate Analysis of Transcript Splicing (MATS) and Differential Exon Usage in RNASeq (DEXSeq) to identify several top candidates, which have upregulated transcript variants during EMT in the PMC42 system. Those top candidates were inspected using SeqMonk to visualize RNASeq reads. Several transcripts were listed on MATS and DEXSeq results as having one of their exon upregulated after EGF treatment in PMC42-LA, including Ladinin-1 (LAD1), Tenascin-C (TNC), Cleft Lip and Palate Transmembrane Protein-1 Like (CLPTM1L), Serine / Arginin-rich Splicing Factor 1 (SRSF1). In this investigation, only LAD1 showed a pronounced up-regulated transcript variant in SeqMonk visualization. Thus, LAD1 is a good candidate as a target for inhibiting EMT in PMC42 breast cancer cell line.