Melbourne Dental School - Theses
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ItemScreening for Type 2 Diabetes Mellitus initiated through the dental setting: a cost-effectiveness analysis( 2019)Background. Diabetes Mellitus (DM) is the fastest growing chronic condition in Australia. Approximately, 30% of DM in Australia is undiagnosed. Early identification may delay or prevent the onset of DM with minimal complication. In the Western Pacific (WP) region, Australia has the highest per capita spending on DM. With the rising cost of healthcare, increasing emphasis is being made to ensure that health interventions are not only practical but also cost-effective that can save resources which otherwise may have to be spent on complication and hospital admission. By stretching the number of contact points between health care providers and individuals seeking care, there is plenty of opportunity for early identification of asymptomatic individuals with Type 2 Diabetes Mellitus (T2DM). With this link between DM and periodontal disease, dentists may have an unrealized opportunity to identify risk groups and refer them to physicians for further care. For any screening activity in the dental setting, the participation of Oral Health Professionals (OHP) is important. Little is known as to how well oral health professionals incorporate into practice on the evidence supporting the link between DM and periodontal disease. Besides that, no previous studies have reported the cost-effectiveness of opportunistic screening using a diabetes risk assessment tool in the dental setting. As such, the aim of the thesis is twofold. To explore the Victorian oral health professionals (OHP) knowledge, attitude and practice (KAP) around DM and to evaluate the overall economic justification of screening for diabetes and pre-diabetes in the dental setting. Methods. A cross-sectional survey of Victorian OHP was conducted. The questionnaire consisted of sociodemographic, practice characteristics and diabetes-related KAP. Descriptive statistics with frequencies and percentages were used to summarize the variables. A Mann-Whitney and Kruskal-Wallis test was performed to determine differences in OHP response to the KAP questions. The screening model consists of a decision tree and a disease progression Markov model to identify the risk of T2DM over a ten-year period. Literature data were used for the risk categorisation and disease transition for health states. The cost-effectiveness of screening was compared to no screening option. A hypothetical population of 40 to 74-year-old Victorian dental patients with no previous history of DM were screened with the Australian type 2 Diabetes Risk Assessment Tool (AUSDRISK). Those identified as high-risk follow-up with the physician for screen diagnosis using Fasting Plasma Glucose (FPG). Based on the previous finding from two-step screening in the dental setting the model made an assumption that 21.5% of the dental patient identified as high risk follow up with the physician. The cost-effectiveness was analysed from a societal perspective. The main outcome measure includes cost per case detected as undiagnosed T2DM, new cases of T2DM. A univariate sensitivity analysis was performed to determine the effect of different physician follow-up rate from the dental setting to identify undiagnosed T2DM. Results. The survey analysis included 197 OHP. General and specialist dentist constitute 65% and 11% of the response and the remainder were dental hygienist and therapist. Around 86% of the OHP showed adequate knowledge of DM. Further 93% and 81% of the OHP expressed positive attitude and practice behaviour towards T2DM screening and management. For OHP to perform chair-side screening for DM, 58% felt it was essential, and 70% felt it was appropriate. More female (67%) and public sector OHP (79%) felt it is important to conduct chair-side screening for T2DM. The majority (65.4%) of the OHP agreed on consent as the most important and insurance coverage as the least important (43%) consideration for T2DM screening. Under model assumption, the number of dental patients identified as undiagnosed T2DM and pre-diabetes were 4,108 (0.3%) and 10,072 (0.8%). The cost incurred for one new case of undiagnosed T2DM and pre-diabetes were AUD 15,508 and AUD 6,325. The Number Needed to Screen (NNS) to identify one new case of undiagnosed T2DM and pre-diabetes were 288 and 117. Among those followed up with the physician, at the end of five years, 81.5% had Normal Glucose Tolerance (NGT), 8.1% had Impaired Fasting Glucose (IFG), 6.9% had T2DM, and the all-cause mortality was 3.5%. At the end of the ten-year period, 10% had T2DM. The overall and disease-free survival was 92.8% and 82.8%. Discussion. Majority of OHPs had adequate knowledge and a positive attitude towards T2DM screening in the dental setting. The survey identified patient willingness as the most important consideration among the OHPs for implementing T2DM screening in the dental setting. The screening model identified several methodological challenges due to incongruent data and unsuitable comparator. Despite that, opportunistic screening with AUSDRISK was found to be neither clinically effective nor cost-effective compared to screening in the medical setting. High screening cost, poor predictive ability of AUSDRISK, low prevalence of the disease, unnecessary physician referral besides uncertain benefits, fear of over diagnosis and poor patient compliance makes screening for T2DM in the dental setting difficult to justify. The model findings are in line with previous estimates on AUSDSRISK as a screening tool. In financially constrained health system resource allocation will need to be based on favourable evidence that screening can reduce disease levels in the community, demonstrate health benefits at an acceptable cost. A two-step opportunistic screening that includes a risk assessment followed by a Point-of-Care (PoC) HbA1c may offer some benefits in the low- and middle-income countries.
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ItemDental implant maintenance and home hygiene – information pathways, clinical practice and patient realities in Australia( 2019)Dental implants have widened treatment paradigms in dentistry since the 1980s and are placed in the millions annually around the world, but plaque-induced peri-implant diseases which can reduce treatment success are not completely understood. The real-life practices of patients and dental practitioners in minimising peri-implant disease risks have not been widely documented. The information sources from which dental practitioners learn peri-implant maintenance are highly variable and also rarely documented. This thesis investigated the flow of information in implant maintenance and hygiene in Australia: from research, to educators, dental practitioners and patients; the limitations present and areas of further development. A series of cross-sectional surveys was conducted to investigate: the hygiene habits of patients with implants in the community, patient-reported outcomes, implant success and peri-implant outcomes; the implant dentistry training attended and provision of implant services by dental practitioners in Australia; dental practitioners’ preferences in implant hygiene instruction, diagnostics and maintenance, including the role of oral health practitioners; and the teaching of implant maintenance topics within implant dentistry education in Australia. A survey of 51 patients in private general dental practice found 7.8% had peri-implantitis and 33.3% had peri-implant mucositis (7.7% and 24.4% of 78 implants respectively). At the implant level, peri-implant disease prevalence was significantly higher where implants were cleaned only with toothbrushing (p<0.001) or had plaque/calculus present (p<0.001). Implant success was significantly reduced if any local factors affecting hygiene accessibility were present (p<0.001). Patients recalled mixed provision of implant hygiene instructions from their treating dentists and reported 7.7% of implants as aesthetically unsatisfactory and 9.0% as having symptoms. A survey of 303 general dentists found continuing professional development was the most common highest level of implant training attended, graduation decade affected the types of implant training attended, and dentists are providing implant treatments increasingly earlier in their careers. Highest attended training level was significantly correlated to greater complexity of implant treatment and maintenance services provided, and a more preventative approach in implant hygiene instruction. Conversely, dentists with little implant training and/or who do not provide implant treatments may not be providing optimum maintenance and preventative information. Compared with the dentists, 154 oral health practitioners surveyed reported more preventative and evidence-based attitudes to implant hygiene instruction, diagnostics and maintenance, and they provided the bulk of preventative services in their workplaces. Implant dentistry education convenors were surveyed (24 respondents outlining 43 programs) and implant maintenance teaching was found to generally reflect the available literature, which is established for diagnostics but limited for patient-performed hygiene, professional maintenance and review. Some respondents acknowledged the need to update their inclusion of implant maintenance topics. As the peri-implant disease, hygiene and maintenance literature develops, current challenges include multi-disciplinary communication and the continuing development of implant dentistry education. By documenting current trends and identifying areas for clinical improvement and further research, it is hoped that this thesis, through the lens of implant hygiene and maintenance, provides possible future pathways for implant dentistry in Australia, to ultimately optimise treatment success and positive patient outcomes.
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ItemThe Role of Candida in Oral Lichen Planus (OLP)( 2019)Purpose: Oral lichen planus (OLP) is a chronic condition characterised by T cell mediated destructions that is currently of unknown cause. OLP can be variably symptomatic with some patients experiencing no symptoms and others requiring extensive symptomatic management. Candida spp. can be found in association with OLP and due to this prophylactic treatment for Candida spp. is usually accounted for in the symptomatic management of OLP. This is despite current evidence not supporting concurrent use of antifungal therapy in the management of OLP with topical steroids. A potential hypothesis for the cause of OLP is an interaction of host genetic susceptibility combined with an environmental trigger that initiates disease in the susceptible host. Another equally likely hypothesis is that OLP is a true autoimmune condition with autoimmunity directed against a currently unknown epithelial autoantigen. The oral cavity represents a unique microenvironment that plays host to many commensal and opportunistic microorganisms. The oral microbiota, specifically Candida spp., could act as an aetiological trigger for the chronic T-cell mediated inflammation the defines OLP, specifically through activation of mucosal associated invariant (MAIT) cells. The role of Candida in the aetiopathogenesis and symptomatic management of OLP is currently unknown. Hypothesis and Aim: The overall hypothesis was that Candida may play an aetiological role in the OLP disease process exerting an effect on T cells and cytokine expression and that adjunctive treatment is required in the symptomatic management of OLP. The overall aim of this study was to determine whether Candida plays an aetiological role in OLP as well as determine if specific treatment of Candida is required in symptomatic patients with OLP. Materials and Methods: 14 control and 7 OLP test patients, 3 assigned to the placebo and 4 assigned to the antifungal treatment group, completed the clinical study. Assessments of clinical appearance, symptoms, Candida, salivary acetaldehyde and medication use were made at 0, 6 and 12 weeks for OLP patients with assessments of Candida and salivary acetaldehyde made at baseline only for controls. 20 random OLP formalin fixed paraffin embedded (FFPE) samples were stained using a fluorescent multiplex immunohistochemistry (mIHC) protocol for the markers cluster of differentiation (CD)3, CD8, DAPI, interleukin 18 receptor 1 (IL18R1), CD161, MR-1 and T cell receptor (TCR) V alpha 7.2. The slides were scanned with the Vectra Automated Multispectral Imaging System (PerkinElmer, USA) to generate multispectral images (MSI). The MSI were then analysed with tissue segmentation and single antibody algorithms for both HALO (Indica Labs, USA) and inForm 2.4.1 (PerkinElmer, USA) to validate a method for quantitative analysis. Following validation of HALO (Indica Labs, USA) for quantitative analysis the above process was repeated on 89 FFPE biopsy tissue samples from 73 patients with OLP (28 asymptomatic, 30 symptomatic and 16 samples with concurrent Candida (9 symptomatic and 7 asymptomatic), for comparison with 15 patient samples of fibroepithelial polyp (FEP). All samples were tested for presence of Candida with periodic acid-Schiff (PAS) staining. A BioPlex assay was performed to measure the cytokines interferon gamma, tumour necrosis factor alpha, interleukin (IL) 17A, IL-18, IL-12p40, IL-12p70, IL-22 and IL-23. Supernatant for this experiment was collected at 8, 12 and 24 hours following prior incubation of peripheral blood mononuclear cells (PBMC) in PBMC media supplemented with either 10% v/v effluent derived from C. albicans biofilms or 10% v/v artificial salivary media (ASM). In addition, some wells were supplemented with either CD28 and/or phorbol 12-myristate 13-acetate (PMA)/Ionomycin. Flow cytometry was performed using TCRV alpha 7.2, CD3, CD161, CD218a, CD4, CD8 and CD45 to define MAIT cells and T cell subsets. Prior to performing flow cytometry PBMC were incubated for 6 hours in PBMC media supplemented with either effluent derived from C. albicans biofilms or 10% v/v ASM with or without CD28. Results: Results of this study showed no significant differences existed between the control group and the OLP test group at baseline with respect levels of salivary acetaldehyde, and Candida colony forming units (CFU). Downward trends were noted in both groups with respect to clinical appearance and subjective analysis of symptoms from baseline to 12 weeks. Trends noted from assessment of CFU and salivary acetylaldehyde levels between the test groups should be viewed with caution due low levels of detection at baseline and the wide spread of data. Minor variability between the tissue segmentation algorithms with the trained algorithm for inForm 2.4.1 (PerkinElmer, USA) being the slightly less variable of the two. For quantitative cell analysis and identification of single antibody positive cells HALO (Indica Labs, USA) proved to be the least variable of the two trained algorithms. The presence of MAIT cell phenotypes were confirmed within the subepithelial infiltrate of OLP. Reduced MAIT cell phenotype expression was noted in the presence of Candida and/or symptoms in OLP with decreased expression of CD161 noted in the presence of symptoms whilst decreased expression of TCRV alpha 7.2 was noted in the presence of Candida. Presence of PMA/Ionomycin and Candida effluent were factors that increased the expression of interferon gamma, tumour necrosis factor alpha, IL-17A, IL-18, IL-22 and IL-23, cytokines that are associated with MAIT cell activation. Across all timepoints the presence of Candida effluent and CD28 resulted in upregulation of IL-18 and tumour necrosis factor alpha. MAIT cells were not significantly affected by the presence of either effluent or CD28 suggesting that neither Candida effluent nor CD28 alone or the combination of the two were shown to induce MAIT cell proliferation. Conclusion: Adjunctive treatment of symptomatic OLP with a topical antifungal did not significantly affect the presence of symptoms, erythema, CFU, Candida spp. or production of salivary acetaldehyde. HALO (Indica Labs, USA) was shown to be the more reliable program for mIHC quantitative cell analysis in FFPE OLP tissue. Analysis of mIHC in OLP FFPE tissue identified MAIT cells within the OLP inflammatory infiltrate with decreased expression of CD161 and TCRV alpha 7.2 noted in the presence of symptoms and Candida respectively. Finally, Candida effluent was unable to induce proliferation of MAIT cells in PBMC. However, cytokines associated MAIT cell activation and OLP, specifically interferon gamma, tumour necrosis factor alpha, IL-17A, IL-18, IL-22 and IL-23, were shown to be upregulated in the presence of Candida effluent derived from C. albicans biofilm.
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ItemThe effectiveness of using a periodontal endoscope as an adjunct to non-surgical periodontal therapy: clinical, radiographic and microbiological results( 2019)ABSTRACT Non-surgical periodontal therapy has been one of the main treatment approaches for managing patients with periodontal disease for decades. The aim of this treatment is to remove bacteria and subgingival deposits, create a “clinically healthy” environment and improve microbial levels to levels that is compatible with health. Conventional non-surgical debridement includes both hand and powered instruments with the ideal end point being a smooth root surface. Periodontal endoscopy was developed in the late 1990s and features miniaturized digital video technology allowing the operator to directly visualise the subgingival environment and at the same time remove any calculus or debris from the root surface with the use of hand or powered instruments. The benefits of direct visualisation technology in improving clinical and inflammatory outcomes were demonstrated in retrospective and prospective studies as well as randomised controlled studies utilizing either split-mouth design or parallel design. However, there was a need to investigate if endoscope scaling and root debridement (SRD) is more effective in reducing levels and numbers of periodontal pathogens as compared to conventional nonsurgical treatment. Also, if bacterial counts decrease, can they be maintained or reduced even further with strict three monthly supportive periodontal maintenance therapy (SPT). Osseous changes can occur after nonsurgical periodontal therapy. However, it is often difficult to determine if changes have occurred due to limitations with conventional radiography. This study included the use of standardized radiographs using customized positioning stents and paralleling x-ray holding devices. Digital radiography and digital software were used to determine areas of osseous change. Aims 1. To compare endoscope-assisted SRD (SRD-with endoscope) to traditional SRD (SRD-only) in reducing clinical parameters over a 12-month period. 2. To assess if endoscope assisted SRD shows more evidence of radiographic changes after the 12-month period compared to conventional SRD. 3. To compare bacterial counts of 11 species including Aggregatibacter(Actinobacillus)actinomycetemcomitans)(A. actinomycetemcomitans); the red complex ((Porphyromonas gingivalis(P. gingivalis); Tannerella forsythia (T. forsythia) and Treponema denticola (T. denticola)); the orange complex ((Prevotella intermedia (P. intermedia); Peptostreptococcus micros (P. micros) ; Fusobacterium nucleatum/periodonticum (F. nucleatum)); the orange-associated complex ((Campylobacter rectus (C. rectus); Eubacterium nodatum (E. nodatum)); the green complex ((Eikenella corrodens (E. corrodens)) and Capnocytophaga species ((Capnocytophaga sp.) (C. sputigena; C. gingivalis; C. ochracea)) in both groups over a 12-month period. 4. To determine the need for surgical intervention in both test and control groups after the 12-month treatment. Materials and Methods The study included 38 participants diagnosed with chronic moderate to advanced periodontitis. Nineteen participants were included in the test group (SRD-with perioscope) and 19 in the control group (SRD-only) by consecutive allocation. Clinical examination included probing pocket depths (PPD), probing attachment levels (PAL), gingival recession, assessment of furcation, mobility, bleeding on probing (BOP) and plaque scores (PI). The measurements were recorded at baseline, three and twelve months and differences in means between groups were calculated for all clinical parameters. The Hain Lifescience Micro-IDent test was utilized for the microbial analyses. Five sterile paper points were inserted into five of the deepest pockets, placed into a sterile test tube and sent in a water-resistant bag to Nehren, Germany for analysis. Eleven putative pathogens namely A. actinomycetemcomitans, P. gingivalis, P. intermedia , T. forsythia, T. denticola, P. micro, F. nucleatum, C. rectus, E. nodatum, E. corrodens and Capnocytophaga sp., were analysed at each time point and compared between the control (SRD-only) and test group (SRD-with perioscope). Analysis of pathogens in their respective complexes namely the red, orange, orange-associated and green complexes were also included. Standardised radiographs were taken at sites with the deepest pockets and in sites displaying angular/vertical bone loss using positioning stents at the baseline and 12-months. The Digimizer Image Analysis software (2005-2018 MedCalc Software bvba, Belgium) was used to measure from the cemento-enamel junction (CEJ) to the alveolar bone crest and changes were tracked in millimetres between radiographs taken pre-SRD and at twelve months. Linear measurements were utilized to determine mean radiographic bone levels (RBL). Results There were no significant differences between groups with regards to age, gender, medical history and disease severity. Both groups showed significant improvements in all clinical parameters after therapy (p<0.05). At three months, no statistically significant differences could be found between groups with mean PPD, mean PAL, BOP and PI. However, for PPDs 7-9 mm the test group had a significantly lower percentage as compared to the control group. At twelve months, the mean PPD was found to be significantly lower in the test group (2.70+0.2 mm) as compared to the control group (2.96+0.4 mm) (p<0.05). In addition, the test group also had lower BOP (4.3+3.2%) percentage as compared to the control group (11.95+7.1%). PI percentage (25.61+3.9%) was also reported to be lower in this group as compared to the control group (30.11+6.3%). The test group had less change in gingival recession (-0.13+0.2 mm) as compared to the control group (-0.5+0.6 mm) from baseline to twelve months (p<0.05). There were no statistically significant differences found between the test and control groups with regards to reduction of periodontal pathogens and their complexes (p>0.05). Both SRD-only and SRD-with perioscope resulted in decreases in numbers of periodontal pathogens including, P. gingivalis, T. forsythia, T. denticola, P. intermedia, P. micro, E. nodatum and E. corrodens post-therapy and at twelve months with numbers remaining below pre-treatment levels. However, A. actinomycetemcomitans, C. rectus, and Capnocytophaga sp. decreased post-therapy in both groups but increased at twelve months. F. nucleatum increased in both groups post-SRD and then were reported at lower levels at twelve months. Both the red and orange complex had lower numbers in the test group at twelve months, with the control group having lower numbers of the green and orange-associated complexes. The mean change in RBL was significantly higher in the test group (0.69+0.3 mm) as compared to the control group (0.49+0.2 mm) (p<0.05). This positive change in mean RBL is indicative of more radiographic bone gain in the test group. There were no differences reported between groups with regards to mean change in RBL for single-rooted teeth (p>0.05). However, for multi-rooted teeth more radiographic bone gain was observed in the test group (0.83+0.5 mm) with a higher mean change in RBL as compared to the control group (0.46+0.4 mm) (p<0.05). The test group had a higher frequency of RBL between 0.5 mm and 1.0 mm and 1.0 mm to 1.5 mm as compared to the control group, inferring that the test group had more sites with radiographic bone gain in this range as compared to the control group. Conclusions Both non-surgical treatment methods used resulted in positive outcomes in clinical, microbiological and radiographic parameters. The adjunctive use of the perioscope significantly improved PPDs 7-9 mm at three and twelve months. The mean PPD at twelve months was significantly lower in the test group as compared to the control group. Less change in gingival recession was observed using the endoscope. The test group had significantly lower BOP% and PI% at twelve months. No significant differences between groups were observed with analyses of the eleven pathogens and complexes of bacteria. The significantly higher mean RBL observed in the test group as compared to the control group is suggestive of more radiographic bone gain in this group. This outcome was also observed for multi-rooted teeth in the test group.
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ItemA study of various factors that influence the success of posterior resin-based restorations( 2019)This thesis is centred on the investigation of several physical properties related to bulk-fill resin composites that may influence the success of posterior resin-based restorations. In addition, a test methodology to investigate the fracture toughness of brittle restorative materials was evaluated, including the potential influence of the storage medium. The thesis work begins with a systematic review comparing the clinical success rates of resin composite restorations in posterior teeth, over two time periods, namely 1995-2005 and 2006-2016. It was shown that the main failure reasons for posterior resin composite restorations changed slightly over these periods, with factors related to the material and tooth becoming more significant in the later period, although recurrent caries remained an important reason for failure. Based on the broad findings of the review, three laboratory-based projects were designed to examine the physical properties of resin-based composites, focusing on the recent bulk-fill restorative materials. The experiments also included the effects of polymerising light-curing units, since the new high irradiance (light intensity) units recommend shorter curing times. In addition, a study on fracture toughness of glass -ionomer cements were conducted to determine whether a simple fracture toughness test would provide useful outcomes for a brittle material such as GIC and whether an ion-containing storage medium such as artificial saliva would influence outcomes. The results of these experiments indicated that variations in material composition and curing light devices had a significant influence on the physical properties such as curing stress shrinkage, the combined temperature of polymerisation and curing light radiant exposure (energy), and wear after thermal ageing. A final experiment investigated the effects of the polymerisation shrinkage on the movement of tooth cusps when the resin composite was exposed to two different irradiances curing light devices. This experiment showed that the high irradiance produced greater and more rapid cuspal movement and a greater rise in temperature within the pulp chamber. The results probably cannot provide a direct translation to the clinical situation. However, it would seem that the use of bulk-fill resin composite materials needs to be managed more carefully than perhaps what has been recommended by manufacturers of such materials.
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ItemThe development of a biodegradable nano-particle vaccine delivery system( 2019)Despite the recent advances in cancer treatment, this disease continues to pose a serious threat to public health. Nanoparticle-based delivery platforms have been shown to be a promising strategy for cancer immunotherapy. Therapeutic cancer vaccines are designed specifically to elicit a potent adaptive immune response, giving rise to the specific eradication of cancer cells. Among the different synthetic nanoparticle carriers currently available calcium phosphate nanoparticles (CaP NPs) are the most promising potential vaccine transporters, and have attracted increasing attention during the past decade. In this thesis, the synthesis of a novel CaP NP vaccine is described and the further investigation of their effects on immune cells is examined. In this study, a novel citrate chelation method for CaP NP synthesis was developed to obtain well-defined, homogeneous NPs. The method of synthesis is innovative, convenient, inexpensive and, most significantly, consistent and reproducible. Additionally, this study is the first investigation to describe the effect of different types of calcium and phosphate salts on NP synthesis. It was found that various sizes of CaP NPs can be obtained by using different calcium and phosphate salts to synthesise the nanoparticles. To further develop the formulation of the nanoparticle vaccine, a layer-by-layer approach to vaccine synthesis was utilised. All experimental parameters of the layer-by-layer approach of nanoparticle formulation were optimised during synthesis to avoid NP aggregation and to increase NP size stability. Significantly, it was found that cross-linking the protein antigen on the NP surface-enhanced salt stability and greatly reduced the host-plasma protein adsorption on to the NP. Moreover, the composition of the protein corona identified by mass spectrometry showed the major component of bound protein was albumin. To study the impact of NP size on the interactions between NPs and host cells three sizes (170 nm, 260 nm and 360 nm) of rod-like shaped NPs were used in vitro, and the effects on epithelial cells and macrophages were observed. This study demonstrated that the three sizes of NPs used in this study can efficiently bind to epithelial cells and migrate through the epithelial barrier and that they induce a cytokine profile from epithelial cells favouring the recruitment of further immune cells. Moreover, the three sizes of cross-linked CaP-PEI-OVA NPs are phagocytosed by RAW 264.7 cells in a dose-dependent manner. Significantly, large CaP NPs induced significantly stronger cell-binding, cellular-uptake, phagocytosis, NF-kappa-B activation, cytokine secretion, and inflammatory cell surface marker expression at the highest NP to cell ratio than the smaller nanoparticles. Finally, a new potential adjuvant with favourable chemical properties for use in vaccine applications was designed and synthesised. A TLR2 ligand, Pam2KK4CG, was synthesised and conjugated to three sizes of calcium phosphate OVA NPs. The effect of these functionalised NPs on macrophages was compared to commercially available Pam3CSK4 and Palmitoyl alone. It was shown that the particles were biocompatible with macrophages and that they were phagocytosed by RAW 264.7 cells in a dose-dependent manner. Additionally, the functionalised NPs significantly increased the expression of cell surface markers (CD40, CD80, and MHC II) in RAW 264.7 cells. Finally, the expression of cytokines was greatly enhanced by Pam2KK4CG lipopeptide treatment as compared to CaP-OVA NPs alone. Taken together, the results presented in this thesis provide an in-depth understanding of the immune response of epithelial cells and macrophages to a synthesised CaP NPs vaccine. Because these cells are vital for the induction of the adaptive immune responses, further work arising from the results of this study may make a significant contribution to the development of therapeutic vaccines for the treatment of cancer.
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ItemImmune Cell Phenotypes in Chronic Periodontitis( 2019)Chronic periodontitis (CP) is a polymicrobial immune-inflammatory disease affecting the supporting structures of a tooth. If left untreated, it leads to eventual tooth loss and loss of function in the oral cavity. The immune-inflammatory component of disease is complex with both the innate and adaptive immune systems involved in disease pathogenesis. Thus, the aim of this study was to phenotype the longitudinal variation in neutrophil and T cell subsets in peripheral blood of chronic periodontitis patients following treatment. Fifty four patients with CP and 40 healthy controls were recruited for the study. Peripheral blood mononuclear cells (PBMCs), saliva and subgingival samples were collected from CP patients at baseline, 3-, 6- and 12-months post-treatment and once from healthy controls. Subjects were assessed by timepoint as well as treatment outcomes. Treatment outcome groups were dependent on the prevalence of sites with PD greater than or equal to 5 mm at the end of the study period where the good treatment outcome group had less than or equal to 10 percent of sites, moderate treatment outcome group had between 10 - 20 percent of sites and the poor treatment outcome group had greater than or equal to 20 percent of sites with PD greater than or equal to 5 mm. The various cells subsets and cytokines were correlated to periodontal parameters. In addition, differences were also assessed between smokers and non-smokers. The periodontal parameters, mean probing depth (PD) and percentage of sites with bleeding on probing (BOP) were increased at all timepoints in CP compared to health and decreased at all subsequent timepoints compared to baseline. Upon separation into treatment outcome groups, patients with poorer treatment outcomes, initially had increased mean PD. In addition, smokers had less BOP at baseline and a decreased response to treatment as seen by a lower reduction in PD at 3-months post-treatment compared to non-smokers. Innate immune responses were examined by assessing surface expression of CD11b, CD16b, CD62L and CD11b. CD62L was associated with treatment-related changes, however, treatment did not affect immature and mature neutrophils proportions. Subsets of immunologically suppressive and normal neutrophils displayed a reciprocal relationship with treatment where suppressive neutrophils decreased and normal neutrophils increased at 3- and 6-months compared to baseline. The red complex bacteria showed a reduction with treatment, however, they were persistently recovered from periodontitis patients at all timepoints in comparison to health. In addition, P. gingivalis was significantly increased in CP compared to health in the poor treatment outcome group. This reduction in red complex bacteria was reflected by a change in the CD4, CD8, CD4+CD8+ and CD4-CD8- T memory cell profile. Naive T cells were decreased in CP at baseline and increased with treatment, while central and effector memory T cells were increased at baseline and decreased with treatment. Poorer treatment outcomes were associated with no changes in the CD4 and CD8 effector memory cells. Examination of cytokine producing effector and regulatory T cell subsets indicated that TCRalphabeta+CD4+ and TCRalphabeta+ cells were the major sources of IFN-gamma and IL-4 in PBMCs, while TCRalphabeta+CD4+ cells were the major cell sources of Foxp3 and IL-17 expressing cells. A reduction in IFN-gamma expressing cells and an increase in Foxp3 expressing cells at 12-months compared to baseline were associated with good treatment outcomes while no changes with treatment were associated with poor treatment outcomes. In vitro expression of IFN-gamma, IL-4, IL-17 and IL-10 upon no stimulation and stimulation with P. gingivalis and concanavalin A was also evaluated in PBMCs. IL-10 was the largest cytokine produced upon P. gingivalis stimulation, followed by IFN-gamma. IFN-gamma and IL-17 expression was significantly decreased in CP at baseline compared to health, while IL-10 and IL-4 levels were not significantly different. In addition, treatment of CP was associated with a reduction in IFN-gamma and IL-4 levels compared to baseline upon P. gingivalis stimulation. Lastly, fifteen T helper 17 cell-related cytokines were evaluated in serum and saliva. IL-1beta, IL-6, sCD40L and TNF-alpha in serum and IL-1beta, IL-6, IL-25 and IL-31 in saliva were significantly increased at baseline compared to health and decreased with treatment. In contrast, serum IL-31 was significantly decreased at baseline compared to health and increased with treatment. In addition, salivary IL-10, IL-17A, IL-17F IL-23, IL-33, IFN-gamma and TNF-alpha also displayed treatment-related reduction. Correlation networks showed that cytokines in saliva showed an increased number of correlations compared to serum. Smoking had an effect on selected immunological subsets. In general, smokers had increased proportions of neutrophils and naive T cells and decreased effector memory T cells and effector memory T cells re-expressing CD45RA. In addition, smokers also displayed aberrant responses in Foxp3 and IL-10 expressing cells in PBMCs as well as IL-1beta and IL-17 cytokine production in serum and saliva. This is a comprehensive longitudinal study of both innate and adaptive changes associated with treatment of CP and compares cell phenotypes across a range of clinical responses to treatment. In particular, this study is the first to report longitudinal variation in suppressive neutrophils, memory T cells, production of four canonical T cell cytokines in PBMCs in response to P. gingivalis and assessment of salivary IL-25, IL-33 and sCD40L as well as IL-31 in serum and saliva in chronic periodontitis. Moreover, this study also showed that good treatment outcomes were associated with treatment-related variation in some memory and T cells subsets, whereas poor treatment outcomes were not. The results from this study provide a preliminary understanding of key modulators of the immune-inflammatory response and furthers our knowledge of the aetiopathogenesis of periodontal disease.
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ItemCharacterization of specific inhibitors of Porphyromonas gingivalis gingipains based on their cognate propeptides( 2019)Porphyromonas gingivalis is a pathogenic bacterium that has a significant role in the progression of chronic periodontal disease. The cell-surface proteases RgpA, RgpB and Kgp collectively known as the gingipains, are major virulence factors of P. gingivalis. Furthermore, P. gingivalis is unable to metabolize sugars for energy production and relies on the gingipains to degrade host proteins to produce small peptides which it uses for nutrition. Thus, strategies that inhibit gingipain function may be of use in reducing the incidence and severity of periodontitis. The Arg-gingipains, RgpA and RgpB and Lys-gingipain Kgp are secreted from P. gingivalis as inactive prodomain-bearing precursors. The amino-terminal propeptides render the proteases inactive until they are cleaved away from the protease at the cell surface. Previously it was demonstrated that when added exogenously recombinant forms of the propeptides of RgpA and RgpB were able to effectively inhibit purified mature high molecular weight RgpA (HRgpA) and RgpB whereas the recombinant Kgp propeptide was a relatively poor inhibitor of Kgp. This present study aimed to examine the inhibitory effect of recombinant gingipain propeptides on the activities of gingipains of P. gingivalis strains isolated from global locales. Furthermore, the effect of the recombinant gingipain propeptides on P. gingivalis growth in biofilm, a clinically relevant state was also examined. The previously developed gingipain protease assays were improved by the addition of optimized concentrations of glycylglycine and cysteine to increase the sensitivity. The assays were then applied to measure the Arg- and Lys-gingipain activities of whole cells and vesicle free culture supernatants (VFSN) of 14 strains of P. gingivalis. It was found that P. gingivalis clinical isolates exhibited different rates of gingipain activities with Vmax of whole cell Arg-gingipains varying 5.8-fold and whole cell Lys-gingipain Vmax varying 2.1-fold across the strains. The P. gingivalis strains also showed more than 100-fold variance in Arg-gingipain activity in the VFSN and more than 350-fold variance in Lys-gingipain activity in the VFSN. Thus, the global P. gingivalis isolates manifest varied Arg- and Lys-gingipain activities. Recombinant RgpA, RgpB, and Kgp propeptides were produced in Escherichia coli (rRgpA-PP, rRgpB-PP, rKgpS16-PP) and added to the gingipain activity assays to determine the inhibitory effects. Using rRgpA-PP, the 50% inhibitory concentration (IC50) for Rgp activity inhibition across the strains ranged from 66 to 227 nM at 95% confidence interval (CI) of 61 to 234 nM. The rRgpA-PP at 670 nM inhibited 87 to 98% Arg- gingipain activity of the P. gingivalis strains except for P. gingivalis 381 that was inhibited 77%. The range of IC50 values of rRgpB-PP against whole cell Arg-gingipain activity were higher than those of rRgpA-PP, with IC50 from 0.69 to 1.8 µM at 95% CI of 0.62 to 1.9 µM, a 5.9 to 11-fold difference depending on the strain. The rRgpB-PP applied at 4.8 µM inhibited 87 to 99% of Arg-gingipain activity of the P. gingivalis, except for strain 381, which was inhibited 76%. Thus, both rRgpA-PP and rRgpB-PP are effective at inhibiting Arg-gingipain activities of the P. gingivalis isolates, but rRgpA-PP is a more effective inhibitor than rRgpB-PP. The inhibitory potential of rKgpS16-PP against whole-cell Kgp activity was minimal, reaching at most 17% against strain W50 when applied at 40 µM. Both rRgpA-PP and rRgpB-PP exhibited a non-competitive type of whole-cell Arg-gingipain inhibition. It was also determined that both rRgpA-PP and rRgpB-PP stored in solution at room temperature for 16 months retained 69% and 64% of inhibitory activity respectively. To discriminate the activities of rRgpA-PP and rRgpB-PP toward the whole cell RgpA or RgpB, rgpA gene mutants of P. gingivalis designated W501 (strain W50 parent) and ECR833 (strain ATCC 33277 parent) and the rgpB gene mutants W50B (W50) and ECR834 (ATCC 33277) were used. Similar Vmax were calculated for all mutants, which were approximately 50% of the activity of parent strains. Propeptide-mediated protease inhibition assay showed that rRgpA-PP and rRgpB-PP could each inhibit both RgpA and RgpB on whole cells. However, rRgpB-PP was 7.8 to 12.5-fold less effective than rRgpA-PP at inhibition of whole-cell Arg-gingipain activity. A method for production of a functional 6 x histidine-tagged RgpA proteinase domain, rRgpAH, was successfully developed and the protein isolated. The kinetics of purified rRgpAH and RgpB were compared. The calculated Km of RgpB for benzoyl-arginine 4-nitroanilide (BApNA) substrate was ~1.4-fold lower than the rRgpAH Km for BApNA, whereas the Vmax of rRgpAH was 2.9-fold higher than the RgpB Vmax. The calculated Ki for RgpA-PP against 5 nM each of rRgpAH and RgpB were similar at 13 nM and 15 nM respectively. The rRgpB-PP Ki at 22 nM and 29 nM against rRgpAH and RgpB respectively was significantly higher (p<0.0001) than the rRgpA-PP Ki. Although rKgpS16-PP was a weak inhibitor of whole-cell Kgp 25% inhibition of 10 nM of purified rKgp with a Ki of 37.5 µM was determined. Biofilm forming capacity of the different P. gingivalis clinical strains was compared following growth in microtiter plates, then measuring biofilm by staining the biomass with crystal violet. It was found that each strain of P. gingivalis produced a different amount of monotypic biomass that could be crystal violet stained. Non-capsular strains 381 and ATCC 33277 were found to produce thick and strongly adherent biofilm biomass. Strains 381, ATCC 33277, 13-1, 15-9, and 7BTORR were used for the biofilm inhibition model, in which increasing concentrations of propeptide were added to the initial bacteria inoculum when seeding the wells for biofilm production. In a dose-response manner, each propeptide inhibited biofilm formation by P. gingivalis with IC50 values for both rRgpA-PP and rRgpB-PP in the low micromolar range, with 95% CI of 1.2-5.2 µM. Most significantly, at the same concentrations, the propeptides also disrupted established biofilm. The rRgpA-PP and rRgpB-PP at 8 and 14 µM, respectively inhibited 99% of biofilm biomass production by P. gingivalis strains ATCC 33277, 381, and 13-1 and 94% of biofilm biomass production by strains 15-9 and 7B TORR. To determine the importance of Arg- and Lys-gingipains in the biofilm formation of P. gingivalis, mutants ECR833 (RgpA null), ECR834 (RgpB null), ECR835 (RgpA/B null) and ABK (RgpA/B and Kgp null) P. gingivalis ATCC 33277 mutants were compared. ECR835 (RgpA/B null) formed significantly higher biofilm biomass than ECR834 (RgpB null) and parent strain ATCC 33277 (P <0.05). The P. gingivalis ABK mutant formed a poorly adherent biofilm with less biomass than the parent ATCC 33277 (P<0.0001). Thus, the absence of RgpA and RgpB resulted in increased biofilm formation by P. gingivalis ATCC 33277, but this effect reversed if Kgp was also absent. The IC50 of rRgpA-PP and rRgpB-PP against P. gingivalis RgpA and RgpB and RgpA/B mutants were found to be higher than the parent strain (P<0.05). However, the IC50 of rRgpA and rRgpB propeptide against the ABK mutant was approximately 6.4 and 8-fold respectively lower than the parent strain. Biofilm forming capabilities of other oral pathogens, Treponema denticola, and Tannerella forsythia, alone and in combination with P. gingivalis ATCC 33277 was assessed. The P. gingivalis ATCC 33277, T. denticola, and T. forsythia cohort formed substantially more biofilm with approximately 39% more biomass than the cumulative biomass of the individual biofilms (P <0.0001). The IC50 against the polymicrobial biofilm for rRgpA-PP was 4.8 ± 0.6 µM and 4.2 ± 0.2 µM for rRgpB-PP. As monospecies, it was found that both T. denticola and T. forsythia formed less biofilm in the in vitro model than P. gingivalis. Nether rRgpA-PP nor rRgpB-PP inhibited biofilm formation by T. denticola. However, unexpectedly, the propeptides did inhibit T. forsythia biofilm formation by 55%. To determine if the propeptides could inhibit biofilm formation by other species rRgpA-PP and rRgpB-PP were applied against Streptococcus sanguinis, E. coli, Chryseobacterium indologenes, and Fusobacterium nucleatum. F. nucleatum biofilm formation was completely inhibited by rRgpA-PP at 12 µM and by rRgpB-PP at 14 µM. However, neither propeptide had any inhibitory effect on E. coli, S. sanguinis or C. indologenes biofilm indicating that the effect of the propeptides is species specific. Analysis of the planktonic phase of the in vitro biofilm model revealed that the growth of P. gingivalis, F. nucleatum, and T. forsythia was inhibited by propeptides, which explained the reduction of biofilm biomass. The spreading of the planktonic phase from the assay wells onto agar plates proved that the propeptide antimicrobial activity was bactericidal. Thus, this study suggests that rRgpA-PP and rRgpB-PP are proteins that have selective antimicrobial activity. The rKgp16S-PP exerted no antimicrobial effect. Porphyromonas gulae is closely related to P. gingivalis, produces Arg-and Lys-gingipains, and is implicated in the etiology of periodontal diseases in companion animals. The kinetics of P. gulae whole-cell and VFSN Arg- and Lys-gingipains were found to be similar to the P. gingivalis 84-3 strain. It was found that rRgpA-PP and rRgpB-PP that were designed using P. gingivalis strain W50 sequences were more effective at inhibiting P. gulae gingipain activity and growth than they were at inhibiting P. gingivalis Arg-gingipain activity and growth. However, rKgp16S-PP did not inhibit P. gulae Lys-gingipain activity. In conclusion, recombinant Arg-gingipain protease propeptides rRgpA-PP and rRgpB-PP are effective at inhibiting Arg-gingipain activities of global P. gingivalis isolates; however, the recombinant Lys-gingipain propeptide was not an effective inhibitor of P. gingivalis Lys-gingipains. Antimicrobial activity of rRgpA-PP and rRgpB-PP was displayed against P. gingivalis and other species with inhibition of planktonic and biofilm growth, but this was not ubiquitous, indicating the mechanism of propeptide antimicrobial action is species specific. The recombinant Rgp propeptides were shown to be stable to room temperature storage. Together the data show that recombinant Rgp propeptides may have the potential for development as inhibitors that impact initiation or progression of periodontitis.
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ItemQuantification of mandibular morphological changes in 3D( 2019)Accurate quantification of mandibular morphological changes is important for orthodontic treatment planning and forensic applications. Traditional two-dimensional (2D) cephalometrics are limited to linear distances or angular measurements, which fail to represent the whole structure of the mandible. Technological advances have made three-dimensional (3D) imaging more accessible. These images successfully preserve the complex mandibular structure and allow clinicians to better understand the growth and development of the mandible. Overall this thesis aims to develop techniques for the analysis of mandibular structure in 3D with particular emphasis on clinical applications.
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ItemBuilding capacity to promote oral health in an Australian community mental health setting( 2018)Half of all Australians will experience a common mental illness during their lifetime. There is strong evidence that people living with mental illness, particularly those living with severe mental illness, experience poor oral health outcomes and face significant challenges in accessing oral health care. Indeed, Australia’s National Oral Health Plan identifies people living with mental illness as a Priority Population with additional and/or specialised oral health needs. Despite this recognition, there is a distinct lack of oral health promotion programs targeting people living with mental illness. The utilisation of non-dental professionals (e.g. aged care professionals, midwives, pharmacists etc.) in promoting oral health has demonstrated considerable success in a variety of settings. However, there is little evidence of this approach involving mental health professionals. Developing oral health promotion skills of mental health professionals may provide an appropriate approach to increase oral health advice and support for people living with mental illness. Therefore, the aim of this PhD project was to investigate how to build capacity to promote oral health in an Australian community mental health setting. All research activities were approved by the Human Research Ethics Committee at the Melbourne Dental School and by the Research and Evaluation Committee at Neami National. A multi-stage sequential mixed methods participatory action research design was used in this project. It was guided by the ‘Framework for Building Capacity to Improve Health’, which is a well-recognised and widely used framework in Australia. Research was conducted within Neami National, one of Australia’s largest community mental health organisations. Stage One explored existing capacity to promote oral health within Neami National through an environmental scan of health promotion action, a post-training evaluation survey after face-to-face oral health training, and a cross-sectional web-based survey of Neami Community Rehabilitation and Support Workers measuring their oral health knowledge, attitudes and professional practices. Stage Two focused on the design, development and implementation of professional development activities to increase capacity of Neami Community Rehabilitation and Support Workers to promote oral health for mental health consumers. This stage involved a multi-state quasi-experimental control group study design, which sought to compare the effectiveness of different training delivery modes (face-to-face, online, blended or none) as part of the ‘Smile for Health’ professional development program. Oral health ‘champions’ in each Neami site were utilised to support the implementation of oral health professional development within their team. Stage Three included process, impact and outcome evaluation, which was used in determining which Smile for Health training delivery mode is most appropriate for use in a community mental health setting. Evaluation activities included a mixed-mode post-training evaluation survey, qualitative focus groups and semi-structured interviews, and a follow-up cross-sectional survey of Neami Community Rehabilitation and Support Workers to measure changes in oral health-related knowledge, attitudes and professional practices. The results of this project demonstrated that any type of professional development can increase the provision of oral health support to mental health consumers. However, it is important to tailor professional development activities to be contextually appropriate. The utilisation of oral health champions was a key enabler in this project, as they had the skills and capacity to problem-solve any issues as they arose and ensured that oral health promotion remained on the agenda within individual teams. Oral health champions offer a sustainable solution to support the implementation of capacity building approaches to increase oral health promotion in community mental health settings. People living with mental illness require specialised oral health promotion programs. This research provides evidence on how to increase capacity to promote oral health in a community mental health setting. Smile for Health offers a contextually appropriate oral health promotion approach for services providing support to people living with mental illness. It is recommended that Smile for Health be rolled out across all Neami sites and potentially implemented in other community mental health services.
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