Melbourne Dental School - Theses
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The development of a biodegradable nano-particle vaccine delivery system
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.
Surgical Management of Recurrent Temporomandibular Joint Dislocation - Systematic Literature Review and Development of a Treatment Protocol
Recurrent TMJ dislocation is a rare entity, clinically distinct from acute or chronic dislocation. It is associated with significant morbidity and deterioration to quality of life for affected patients. Recurrent temporomandibular joint (TMJ) dislocation can be challenging to treat and current understanding regarding aetiology and management of this condition is limited. The aim of this thesis was twofold. The first was to conduct a systematic review regarding the current understanding of managing recurrent TMJ dislocation. The second aim was to review the surgical management and long-term outcomes of patients with recurrent TMJ dislocation who presented to a single Hospital Department over a period of six years so as to formulate a practical treatment algorithm. A retrospective review of cases surgically managed for recurrent TMJ dislocation was undertaken with respect to patient demographics, clinical features, surgery provided, and long term follow up. A literature review was conducted using PRISMA guidelines to identify papers published between 2006 and 2016. The resultant papers were analysed. A total of 33 papers were found relevant to the study. Minimally invasive techniques described included autologous blood injection, which was associated with an overall success of 80% at 12 months. Other modalities investigated included OK-432 sclerotherapy, laser capsulorrhaphy, botulinum toxin of the lateral pterygoid muscle or modified dextrose. These publications show promising success rates. Surgical techniques described included disc plication, eminoplasty and eminectomy. These modalities had a similar success rate, although numbers were limited. For the second part of this thesis, a total of 14 patients were identified who were managed for recurrent TMJ dislocation over a 6-year period from 2010 to 2016. The cases were followed up for a minimum of 12 months and a maximum of seven years. Results showed effective long-term resolution of symptoms using a combination of eminectomy, disc plication (meniscopexy) and where clinically indicated, lateral pterygoid myotomy. This thesis found that the true incidence of recurrent TMJ dislocation is unknown and aetiology is limited to expert opinion. The current understanding of management for recurrent TMJ dislocation is limited to case series and case reports. This thesis compiles the current understanding of management of recurrent TMJ dislocation. A decision making algorithm, with a personalised, step-wise approach to treatment is presented. The retrospective review portion of the thesis has shown that a combination of eminectomy and disc plication (meniscopexy) is effective in providing long term positive outcomes in the surgical management of recurrent TMJ dislocation. Those cases of recurrent TMJ dislocation resulting from dystonia of the lateral pterygoid muscle also benefitted from additional lateral pterygoid myotomy.
Immune Cell Phenotypes in Chronic Periodontitis
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.
Exploring the oral health curriculum in Australian pharmacy schools
Introduction Poor oral health continues to be prevalent in Australia despite ongoing advancements in oral health knowledge and care. Without innovative strategies to improve the oral health of the population, the quality of life for an increasing number of Australians will be negatively affected as poor oral health extends beyond the mouth and can affect general health and well-being. Beyond the dental clinic setting, pharmacists have been recognised in the literature to have an important role in oral health care. The potential to expand the role of pharmacists as oral health advisors has also been acknowledged. While previous studies explored the knowledge and opinions of pharmacists regarding oral health, no research has been completed to explore the extent of oral health content that is currently included in Australian pharmacy schools’ curricula or on the knowledge and opinions of the pharmacy students who are about to graduate as health professionals. Aim The aim of this study was to investigate the knowledge, attitudes and perceptions towards the role of pharmacists in oral health among final year pharmacy students in Australia, and to investigate the extent of the oral health content in Australian pharmacy curricula. Methods A cross sectional study of pharmacy students across 8 Australian pharmacy courses was undertaken using an anonymous online survey. In addition, semi-structured interviews were conducted with pharmacy course coordinators or convenors to discuss the oral health content in their course curricula. Survey results were analysed using SPSS software (SPSS 25.0, Chicago Il, USA) and the findings summarised using descriptive statistics. Phone interviews were recorded, transcribed verbatim and analysed thematically. Results A total of 45 pharmacy students across the nation completed the online survey. Almost half of the students (48.9%) reported that oral health was not included in their course. Many believed that pharmacists have an important role in oral healthcare, however only 38.9% perceived that pharmacists were appropriately trained to provide oral health education. Most students (91.7%) believed that professional relationships between pharmacists and dental practitioners could be improved, and that pharmacists had the potential to be more involved with preventing oral health issues (86.1%). Three main themes emerged from the course convenor interview study: (1) That pharmacists have a role in oral healthcare, (2) That oral health is being taught in pharmacy courses, however each did so in a varied manner, (3) Lack of space in course curricula is the key barrier for further inclusion of oral health care content in pharmacy courses. Conclusion Overall, the findings of this study provide evidence that the oral health content in pharmacy curricula in Australia is inconsistent, with students indicating that they wanted more education on oral health topics. Both students and course convenors recognised that pharmacists have an important role in oral healthcare. Therefore, pharmacy courses in Australia should consider expanding the coverage of oral health content to provide graduates with the confidence and skills they need to improve the oral health of the community.
Screening for Type 2 Diabetes Mellitus initiated through the dental setting: a cost-effectiveness analysis
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.
Characterization of specific inhibitors of Porphyromonas gingivalis gingipains based on their cognate propeptides
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.
Quantification of mandibular morphological changes in 3D
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.
Building capacity to promote oral health in an Australian community mental health setting
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.
The effect of Treponema denticola on Porphyromonas gingivalis phenotypes and transcriptome
Chronic periodontitis is an inflammatory, bacterial biofilm-associated disease resulting in destruction of the tooth’s supporting tissues. The imminent progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Living in a complex oral polymicrobial community, these two bacterial species display close association via physical interaction and metabolic cooperativity in the biosynthesis and cross-feeding of growth substrates. These interspecies interactions result in the coordination of their physiological activities, some of which exhibit complementary and combinatory effects in enhancing their growth and virulence factors. A previous study demonstrated that coculture of T. denticola and P. gingivalis in a continuous system led to upregulation of T. denticola glycine utilisation systems. Likewise, P. gingivalis increased the production of free glycine by proteolytic hydrolysis of peptide-bound glycine during growth in T. denticola conditioned medium (TdCM), suggesting cross-feeding of glycine from P. gingivalis to T. denticola. Free glycine is an important nutrient source for T. denticola, contributing to a dramatic increase in T. denticola growth rate and biomass. Given that P. gingivalis glycine release was stimulated in TdCM, this study aimed to determine and characterise the T. denticola stimulatory factors that had been released into TdCM. TdCM was fractionated by size filtration and reversed-phase high-performance liquid chromatography (RP-HPLC) to determine the most active stimulatory fractions. The free glycine produced by P. gingivalis in different TdCM fractions was then quantified using a glycine enzyme-linked immunosorbent assay (ELISA) kit and liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS). As several RP-HPLC fractions of TdCM appeared to stimulate the release of free glycine by P. gingivalis, this indicated that there was no specific signalling molecule that stimulated P. gingivalis to produce glycine. Instead, the release of glycine by P. gingivalis was likely due to P. gingivalis proteinases that further digested the peptides that had been partially processed by T. denticola proteases during growth in the medium. Candidate peptidases of P. gingivalis that could be involved in the hydrolysis of glycine-containing peptides into free glycine were selected by bioinformatic predictions of the localisation and specificities of P. gingivalis putative peptidases. Interestingly, inactivation of PG0753 and PG1788 that encode putative PrtQ collagenase and C1 family cysteine peptidase respectively, showed a reduction in the rate of free glycine production relative to P. gingivalis calculated cell number in OB:CM, relative to wild type. These results indicated that these two peptidases might play a role in the release of free glycine by P. gingivalis in the presence of T. denticola stimulatory factors. Other potential multimodal interactions of T. denticola and P. gingivalis were examined by investigating the differential gene expression profiles of P. gingivalis during growth in TdCM, compared to oral bacterial growth medium (OBGM). There were a total of 132 genes that showed differential expression, which included transcripts related to metabolic pathways, signal transduction systems, transcription regulatory systems, nucleic acid interacting protein encoding genes, transporters and hypothetical protein encoding genes. This work generated more testable hypotheses of the molecular effects of T. denticola on P. gingivalis gene expression and as well as potential mechanisms that might contribute to P. gingivalis and T. denticola physiological interactions.
Oral cancer prediction using microRNA in oral swirls
Purpose: To study a clinically convenient sample type, oral swirls as a source of microRNA for analysis in oral disease states. Objectives: To study a panel of OSCC-associated microRNA, identified in next generation sequencing (NGS) data of tissue specimens, in oral swirls from individuals with OSCC and oral potentially malignant disorders (OPMDs). Materials and Methods: Oral swirls were inspected using electron microscopy and tested for robustness by challenge with RNase and temperature shifts and analysis using microRNA by qPCR. Oral swirls were collected from 190 individuals with and without oral mucosal conditions. An OSCC- associated panel of microRNAs was identified in FFPE specimen NGS data and a fresh frozen specimen data set from The Cancer Genome Atlas. This panel was studied by qPCR in the oral swirls from 190 individuals with and without mucosal abnormalities including OSCC (n=53) and OPMDs (n=74). Results: Oral swirl sourced microRNA was consistently detected and demographics, comorbidities and oral disease states did not affect the yield of RNA. A reproducible workflow was used to extract RNA from oral swirls collected from 190 individuals. Upregulation of miR-31, miR-21 and downregulation of miR-99a, let-7c, miR-125b and miR-100 was found between OSCC and controls in NGS data of both FFPE and fresh frozen specimens. These microRNAs were studied in a training set of 15 OSCC vs 15 control oral swirls to develop a cumulative dysregulation score (AUC 0.95 (95% CI, 0.88-1.03)) and categorical algorithm-determined risk category. Utilizing the presence of HIGH-risk in 53 OSCC vs 54 controls, the test was 86.8% sensitive and 81.5% specific. One case of malignant transformation within the OPMD cohort demonstrated longitudinal utility of the test. Conclusion: Oral swirls provide a clinically convenient sample type for assessment of microRNA in disease states. This is first study to analyze microRNA sourced from oral swirls from individuals with and without mucosal abnormalities including OSCC and OPMDs. A HIGH-risk dysregulation signature was found to be accurate in indicating the presence of OSCC and exampled to parallel malignant transformation. Assessment in further longitudinal studies is warranted.
Early childhood caries in Cambodia
Children in Cambodia have a severe burden of caries and data from the Cambodian national oral health survey reflect that four in five 6-year-old children have one or more pulpally involved teeth. One of the key features of dental caries in Cambodia is that most of the lesions go untreated and most of the dental services are orientated towards those in an urban setting and involve management of pain only. Global evidence on prevention of dental caries suggests that interventions focusing on topical application of fluorides in the form of toothpastes or varnishes and application of pit and fissure sealants (FS) might be the most effective interventions for reducing caries experience. Two groups of investigations were conducted; the first group of investigations were part of the SEAL Cambodia project and the second group were part of the Cambodia Smile Project. The aims of the SEAL Cambodia project were to evaluate two different protocols for the placement of glass ionomer cement-based fissure sealants and the prevention of dental caries in the FPM of 6 to 8 year old children in three provinces of Cambodia, then to investigate the caries preventive effect of a refined protocol for GIC fissure protection in FPM of 6 to 8 year-old children in Cambodia. The Cambodia Smile investigation aimed to describe the epidemiological aspects of Early Childhood Caries in a Cambodian context, then investigate the effectiveness of a pilot strategy for the reduction of dental caries experience in Cambodian preschool children through an integrated primary health care model. The original SEAL Cambodia project rendered a non-significant 10% preventive fraction for new carious lesions on first permanent molars after 1-year. The modified protocol rendered a 90% preventive fraction at 1-year and 30% preventive fraction at 2-years. The Cambodia Smile results affirmed existing evidence that Early Childhood Caries (ECC) affected a large proportion of young children and that a package of oral health interventions integrated with the routine vaccination schedule can render a 66% reduction in the severity of dental caries among 2-year-old children. This collection of studies represents the first efforts to build evidence around the reorientation of dental services towards preventive therapies in a Cambodian context. Further investigation is needed to better understand the social, structural and behavioural aspects of the ECC phenomenon in Cambodia in order to better inform strategy. The SEAL Cambodia project provides sufficient evidence for an appropriate clinical protocol for the application of FS in a Cambodian context. The Cambodia Smile pilot provides sufficient evidence to justify up-scale and monitoring of a similar project to reduce the disease burden among a larger proportion of Cambodian children.
The rectangular block implant: a novel implant design for the posterior atrophic ridge.
A horizontally oriented rectangular block implant (RBI) is proposed: - maximizing the use of the resorbed posterior ridge length and qualifying as a “short” implant, with a surface area equivalent to that of longer cylindrical counterparts. The aims of this qualitative study were firstly, to design and manufacture the RBI and biomechanically test the RBI-abutment componentry. Secondly, to surface treat the RBI and validate its surgical placement protocol. Thirdly, to assess the osseo-integration of the RBI and finally, to biomechanically test the osseo- integrated RBI-bone environment. RBIs and abutments, were manufactured through the use of lathe turning at a tolerance of 10 m. Assembly was assessed with micro-computerized tomography (-CT). A modified ISO 14801 cyclic fatigue protocol of epoxy embedded implant-abutment-crown complexes was used until failure (> 2000 N). Post-fatigue analysis was performed with -CT. The peri-implant embedding medium strains were assessed using surface strain gauges: - buccal strains were significantly higher (p<0.01) than approximal strains for all force levels. RBI surface preparation was performed with Al2O3 grit blasting followed by acid immersion. Mean surface roughness (Sa) assessments were determined using laser profilometry. 250 m grit blasting at 1300 kPa, at 20-30 mm from and 900 to the surface, for up-to 5 minutes, followed by acid bath (0.01M HCl, 800C, 18 hours) immersion, was capable of consistently producing Sa values in the range of 2.5-3.5 m. Scanning electron microscopy (SEM) highlighted surface sintered and layered Al2O3 particles. Piezo surgery and press-fit surgical protocols were used in the placement of 12 RBIs into greyhound dog posterior mandibles. These protocols proved to be an effective method for the surgical placement of the RBI. Osseo-integration was assessed at 12 weeks with resonance frequency analysis and torque tests. One RBI failed to integrate. Bone to implant contact (BIC) was assessed using -CT and SEM, which yielded a mean percentage level of 46.9 ± 5.3 %. The biomechanical assessment of the osseo-integration was tested through paired linear force applications: pull-out, lingual-buccal push and mesial-distal push. Pull-out tests yielded 695 N and 712 N (equating to a shear strength of ~10.6 MPa). Buccal push test results yielded failure at 489 N and 500 N, while distal push test results yielded failure at 800 N and 930 N. Overall, the osseo-integrated RBI environment was capable of exceeding the corresponding human masticatory maximal axial and non-axial force vectors. The distribution and magnitude of the strains in the loaded peri-implant cortical bone area were assessed with cortical strain gauge analysis of a cyclically 300-off-axis loaded crown-abutment-implant complex. The result was a complex picture of a bucco-lingual horizontal tension- compression, a bucco-lingual vertical torsion and a mesio-distal rotatory torsion: - indicative of horizontal and vertical torsional flexure of the mandible. Physiological bone strain limits were not exceeded, even at 1000 N. This was partly a function of the block’s flat faces, more evenly and efficiently distributing the applied stresses to the osseous interface. Further studies are required to clarify the bases of these findings.