The overall aim of this thesis is to examine the underlying physical basis of dentinal hypersensitivity and to assess methods of treating this cause using imaging techniques. The scanned probe microscopy (SPM) techniques are then extended to the study of carbon-based electrode surfaces, as described in the final chapter. The use of scanning electrochemical microscopy (SECM), combined with in situ pressure-time measurements, is described as a means to investigate the flow of fluid through human and bovine dentine, and the subsequent effect of occlusion treatments on this flow. Scanning Ion Conductance Microscopy (SICM) is also introduced as a technique for imaging dentine, with instrument design and development described, and also calibration of the technique. Laser scanning confocal microscopy (LSCM) coupled to a constant volume flowpressure measuring system is introduced as a new technique for the quantitative measurement of fluid flow across porous materials. The methodology described herein firstly allows a ready assessment of the general efficacy of treatments via hydraulic permeability measurements. Second, LSCM images allow the nature of the flow process and the mode of action of the treatments to be revealed at high spatial resolution. For the particular case of dentine, we demonstrate how the method allows candidate treatments to be compared and assessed. To complement the studies into dentinal hypersensitivity, microscopic dissolution of bovine enamel is investigated. This chapter describes a novel approach, based on SECM, to promote the localised dissolution of bovine enamel, effected by the application of a proton flux to the enamel surface from a UME positioned within 5 μm of the surface, in aqueous solution. The approach results in a well-defined “acid challenge” yielding well-defined etch pits that were characterised using light microscopy and white light interferometry. The effect of etching in the presence of lactate is considered, as is the effect of treating the enamel samples with sodium fluoride prior to etching. The approach described is amenable to mass transport modelling, allowing quantitative interpretation of etch features. The techniques developed throughout the thesis are applied to the investigation of two types of carbon electrodes: boron-doped diamond (BDD) and highly ordered pyrolytic graphite (HOPG). Heterogeneities in the electroactivity of these substrates are explored.A scanning micropipet contact method (SMCM) is described which promises wide-ranging application in imaging and quantifying electrode activity at high spatial resolution.

Aim: To evaluate the effect of various environmental (clinical) conditions on the physical and chemical characteristics of Mineral Trioxide Aggregate (MTA). Methodology: Initially preparation of specimens was standardised. Moreover, a novel mixing technique, trituration of encapsulated MTA, was developed. The effects of acid and blood contamination on various characteristics of MTA including compressive strength, surface microhardness, push-out bond strength and total porosity were then evaluated. Furthermore, by using X-ray diffraction analysis the hydration process of blood contaminated MTA was studied. In addition, the microstructure of contaminated MTA specimens was compared with control groups. Results: Methods of mixing and placing MTA significantly affected the hydration process and consequently the physical properties of the material. The lowest and greatest compressive strength, Vickers surface microhardness, and push-out strength values of MTA were found after exposure to pH levels of 4.4 and 7.4, respectively. In addition, scanning electron microscopy revealed a lack of needle-like crystals when the material was in contact with more acidic solutions. The hydration state of specimens partially mixed with blood was more complete than those mixed entirely with blood and less than specimens that were hydrated only with water. Conclusion: In experimental investigations, use of controlled mixing and placement techniques when using MTA is essential in order to standardise specimen preparation. Delaying the placement of the final coronal restoration in clinical situations when MTA is contaminated is recommended so that the material can acquire sufficient physical properties to withstand the acid-etch procedure and the condensation pressures that occur during the placement of a restoration and/or produced through indirect masticatory forces.

Dental pulp stem cells (DPSCs) play an integral role during dentine injury where they migrate towards the injury site, proliferate, differentiate into odontoblast-like cells and secrete a mineralised matrix, protecting the vital pulp tissue and preserving the tooth organ. Dentine matrix proteins (DMP) may have a role in stimulating this reparative dentine formation. Multiple adult stem cells make up the DPSC population. Within this study progenitor cells were isolated from dental pulp by preferential fibronectin adherence (FNA) or using magnetic activated cell sorting (MACS) to isolate P75 expressing neural crest cells. Clonally expanded colonies were established. The proliferative FNA and P75-sorted populations were shown to be distinct progenitors with differing morphology and stem cell marker expression. One FNA clonal population was differentiated into mesenchymal lineages that established it as a multipotent DPSC population. Clonal populations were supplemented with DMP in vitro to examine the potential role of DMP in modulating cell behaviour during dentine injury. Supplementing with DMP had a dose dependent response on DPSC viability, increased cell numbers,reduced apoptosis and promoted cell migration, suggesting that the growth factors in DMP may have a positive synergistic effect on DPSC behaviour. Fluorescently stained DPSCs were microinjected into transverse tooth slices and placed in culture to develop an ex vivo model to study DPSC behaviour in a tooth environment. Injected cells remained viable after 7 days of culture, providing proof of concept data that DPSCs can be localised for study in situ. This thesis was successful in the isolation of clonal populations representing progenitor cells with different characteristics. Supplementing DPSCs with DMP promoted cell behaviour facilitating reparative dentinogenesis, indicating a potential clinical application for DMP in restorative therapy. The study of DPSCs using the ex vivo model will be important in further development of these novel therapies for dental tissue regeneration.

The four individual phenotypic cell populations persisted in culture. On recombination into the organotypic system there were marked growth variations that may be attributable to diverse cytokine/receptor levels involved in epithelial-mesenchymal interactions. Such varying cytokine levels may represent intrinsic differences in sub-sets of the population that are at greater or lesser risk of developing periodontal disease.

Conscious Sedation is a pharmacological intervention which enables anxious patients to have dental treatment. Although there is a strong research tradition into the efficacy of sedation modalities, there is a weak evidence base for the experience of sedation by those who use it. The aim of this thesis was to explore patients’, referrers’ and providers’understandings and experiences of conscious sedation and the Secondary Care Sedation Clinics which use it. A qualitative study was undertaken of stakeholders’ experiences of conscious sedation provided by five Secondary Care Sedation Clinics within the United Kingdom. Data were collected through thirty one semi-structured interviews, which were transcribed verbatim and subsequently analysed using a constant comparative method. The data show that sedation and secondary care sedation clinics are imbued with a variety of interpretations by differing participants. Patients primarily perceive sedation clinics as access points for addressing dental needs, whilst clinicians also anticipate an influential role in rehabilitating patients to primary care and recognise the importance of such settings for training future dentists. Successful sedation provision requires a variety of work, and sedation clinics play a hosting role to visiting patients as hinterlands to the dental world. The outcome of patients’engagement with sedation clinics varies from breaking avoidant patterns to creating a cycle of sedation dependency, whilst the process of sedation performance has a potential negative impact for providers. 2 The purposes and processes reported by participants mirror those of Victorian domestic parlours. This thesis suggests a novel theoretical construct of clinical ‘Parlours’. Such frontier social structures provide safe interaction for patients in a temporarily hosting environment. They require front-stage performance augmented by back-stage work, and seek to influence patients in the long-term whilst providing short-term clinical services. Further research is required to explore the transferability of such a concept to other clinical settings.

Introduction: Several observational studies support an association between periodontal disease and Alzheimer’s disease (AD). Poorly managed oral hygiene together with the immunosuppressed status of demented patients appears central to this hypothesis as together they contribute not only to an increased incidence of oral infections but also to recurrent bacteraemia that can seed oral bacteria into systemic circulation. The aim of this study was to establish a link between periodontal disease and AD with a view to identifying the red complex periodontal disease bacteria (Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis) and/or bacterial components in human AD and non-AD brain tissue and explore the proof of concept of the red complex bacteria accessing the brain of ApoEnull mice during experimental periodontitis, and assess how they may contribute to the development of AD pathology. Methods: Molecular techniques (PCR, cloning and sequencing) were employed for investigating the presence of bacterial DNA within the specimens (human or mouse) using two different approaches (universal and species specific primers). The presence of specific virulence factors were determined using anti-bacterial antibodies. The innate immune responses were detected using antibodies against complement activation, alongside inflammatory assessment using specific antibodies for activated microglia and astrocytes. Further, histology staining was used to assess tissue preservation and the presence of pathological hallmarks of AD. Results: Human AD and non-AD controls failed to demonstrate the presence of red complex pathogens when analysed using molecular methodologies. However, immunofluorescence labelling of a virulence factor (LPS) was positive for P. gingivalis in 4 out of 10 AD cases. Immunoblotting demonstrated bands corresponding to P. ginigivalis LPS in the same AD brain specimens (p = 0.029). Analysis of brain tissue from ApoEnull mice induced with periodontal disease using molecular methods demonstrated 6 out of 12 ApoEnull mice brains contained P. gingivalis genomic DNA at 12 weeks (P = 0.006), and increased to 9 out of 12 at 24 weeks (P = 0.0001). In addition, tissue sections of infected mice demonstrated periodic acid-Schiff (PAS)-positive, argyrophilic inclusions in the hippocampus at both time points, which also labelled positive with the bacterial-specific anti-peptidoglycan antibody. Also, it was noted that microglia in both infected and control groups demonstrated strong intracellular labelling with C3 and C9, presumed on-going biosynthesis, however, the pyramidal neurons of the hippocampus in 4 out of 12 P. gingivalis infected mice brains were clearly opsonised with C3 activation fragments (P = 0.032) suggesting they were under attack from complement mediated lysis. Conclusion: These results show P. gingivalis was able to access the brain of humans and ApoEnull mice, supporting the concept of the focal infection theory. Together these results suggest a potential link with AD via the periodontal pathogen translocating from its original oral niche to the brain. ApoEnull mice induced with periodontal disease demonstrated the intracerebaral innate immune responses were initiated by local CNS cells, which not only contributed to a higher inflammatory burden but also bystander damage of functional neurons in the hippocampus area of the brain which is associated with memory. Although further research is needed to establish clinical measures that demonstrate a cause and effect relationship between oral infections and AD, this study does provide initial support to the role of periodontal pathogens in the development of dementia. Early treatment of periodontal disease in addition to greater awareness of the importance of maintaining good oral health may halt or slow down the progression of this debilitating disease.

The successful outcome from implant procedure relies heavily on the integration between the implant and the surrounding bone tissues. Besides, Type 2 Diabetes Mellitus (T2DM), which is linked with delay of osseointegration and reduction of bone-implant interface, further compromises the success rate of implant in diabetic patients. Apart from hyperglycaemia, the precise mechanism of diabetes influence on bone repair associated with dental implants is not completely understood. Nevertheless, the transforming growth factor-β1 (TGF-β1) has been indicated to increase healing processes, by exerting the stimulatory role on mesenchymal stem cells (MSCs) and macrophage populations during the inflammatory stage of bone repair. Moreover, the bioavailability of growth factors has been associated with the functional role of SLRPs, particularly biglycan and decorin. However, the responsiveness of each relevant bone-repair cell and biomolecule during bone repair in a diabetic environment has not been fully evaluated. On the other hand, the in vivo osseointegration of implant in T2DM animal models,investigated in respect to the expression of TGF-β1 by MSCs, demonstrated statistically significant differences in TGF-β1 labelled between the young diabetic and the control groups. Besides, the in vitro assessment demonstrated alterations for TGF-β1 expression and synthesis by osteoprogenitor cells, macrophages populations,between cells with different proliferative states, and prior hyperglycaemic-induction. Moreover, hyperglycaemia altered osteogenic and adipogenic differentiation capacities in MSCs. The data also suggested that hyperglycaemia induced lower proliferative capacity in MSCs, which led to significant changes in growth factor and proteoglycans bioactivity in bone repair. Hence, the data gathered from both in vivo and in vitro experiments suggested the potential association of MSCs proliferative stage with bioavailability of TGF-β1 and proteoglycans sequestration in the extracellular matrix compartment. Apart from that, the inter-dependent relationship observed between the osseointegration biomolecules directly exerted a synergical impact on the capability of MSCs to form osteoblast and further stimulate bone formation in order to induce bone-healing processes. Thus, the original contribution of this study to the field of reparative medicine is the novel identification and the characterisation of key biological components in both cellular and molecular bone repairs; the osteoprogenitor cell populations, as well as the macrophages, in relation to hyperglycaemia that directly influences growth factors, signalling the role of proteoglycans during the bone repair processes in T2DM. Collectively, the evidence gathered within this study is highly valuable to assist in elucidating the relevant therapeutic target to accelerate bone repair processes in T2DM patients. Keywords: diabetes, osseointegration, hyperglycaemia, bone, growth factors.

The dentine-pulp complex is a unique, dynamic organ capable of responding to mild and aggressive injurious stimuli. Deep caries or tooth fractures that extend close to the pulp can lead to inflammation and ultimate pulpal necrosis if not managed appropriately. Current therapeutic regimes for such scenarios include the use of vital pulp (capping) treatments, which attempt to stimulate the pulp’s auto-repair capacity which is still poorly understood and consequently not exploited to its full potential. Amongst the possible mechanisms involved in inducing a dentinogenic response in vital pulp therapy, the ability of dental materials in solubilising cell signalling molecules from dentine has been proposed recently. As such, the role of growth factors belonging to TGF-β superfamily in particular has been highlighted during dentine repair and regeneration (Graham et al. 2006). In recent times clinical practice has moved towards the use of dentine bonding agents and composite restorations even in deep dentinal cavities that extend close to the pulp in the hope of reducing the effects of microleakage, amongst others (Lynch 2008). However, although such treatments appear moderately successful in practice, the justifications for introducing new protocols to improve dental pulp protection and dentine bridge formation are not currently driven by biological knowledge. This in vitro study investigated the effects of dentine conditioning by several clinically used etching agents/ dentine conditioners (components of adhesive restorative systems) on the solubilisation of bioactive dentine matrix components and dentine regeneration (by assessing the impact on DPSC phenotype and behaviour). It was found that growth factors (TGF-β1, BMP2 and VEGF) were exposed and released following dentine treatment by EDTA, phosphoric and citric acid. Polyarcrylic acid on the other hand was ineffective in solubilising dentine matrix proteins. Dentine surface conditioning by EDTA, phosphoric and citric acid resulted in an up regulation in the expression of genes associated with osteoblast/odontoblast-like cell differentiation (OPN and ALP) in DPSCs, however surfaces treated with phosphoric acid appeared significantly less supportive of cell growth following 8 days in culture. These findings provide direction for future research and take crucial steps for informing clinical practice as to the choice of materials and techniques in vital pulp treatments.

Objectives: To investigate the remineralisation of enamel subsurface lesions with CPP-ACP toothpaste compared with regular fluoride toothpaste in orthodontic patients. Design: Randomised controlled clinical trial with a cross-over design. Setting: Orthodontic department at the Liverpool University Dental Hospital. Participants: 12 orthodontic patients receiving fixed orthodontic treatment. Methods: Demineralised sub-surface enamel lesions were placed in situ onto a fixed appliance in the lower premolar region. Participants were randomly allocated to receive either standard fluoride toothpaste or a CPP-ACP paste (GC Tooth Mousse™) in addition to the fluoride toothpaste. Transverse Microradiography (TMR) was used to analyse and compare the mineral content profiles of the lesions. Results: Mineral loss ΔZ reduced by 15.4% and 24.6% for the fluoride and CPP-ACP groups respectively, with a statistical significant difference between these groups (p = 0.023). Lesion depth reduced by 1.6% and 11.1% for fluoride and CPP-ACP groups respectively, with a statistically significant difference between these groups (p = 0.037). Lesion width reduced by 4.5% and 15.3% for fluoride and CPP-ACP groups respectively, with a statistically significant difference between these groups (p=0.015). Conclusion/Implications: Remineralisation occurred during the in situ phase regardless of treatment group allocation, however the CPP-ACP combined with fluoride group demonstrated a statistically significant increased remineralising effect. CPP-ACP paste may be beneficial for patients undergoing orthodontic treatment who are at high risk of demineralisation or who have demonstrated early signs of white spot lesion formation. Trial Registration: Registered on Current Control Trials http://www.controlled-trials.com/ISRCTN04899524.

Aims: 2005-2007 abstracts This study aimed to identify the: • number of clinical trials that were presented from 2005-2007 at the conferences of the: o American Association of Orthodontists (AAO), o European Orthodontic Society (EOS), o International Association for Dental Research (IADR), o European Organisation for Caries Research (ORCA) o Australian Society of Orthodontists Congress (ASO) • abstracts that went on to be published as a full paper in a peer reviewed journal. • time to publication for those abstracts that were subsequently published as a full paper in a peer reviewed journal. • following characteristics of the abstract and determine their influence on the rate of and time to publication: o Result significance: (Significant, Non-significant, or Unclear) o Mode of presentation (Oral or Poster) o Study design (RCT / CCT) o Sample Size: (Absolute number) o Funding disclosure: (Yes / No) o Continent of origin: (North America, South America, Europe, UK, Asia, Africa, Australasia). o Primary author: • Gender (Male / Female / Unclear) • Professional status: (Professor / Non-professor / Unclear) • Identify reasons why abstracts did not achieve publication. University Teachers Group (UTG) abstracts This study aimed to identify the: • number of abstracts presented at the University Teachers Group session, from 1999-2010, at the British Orthodontic Conference. • following characteristics of the abstract and determine their influence on rate of and time to publication: o Funding disclosure: (Yes / No) o Dental School of origin • abstracts that went on to be published as a full paper in a peer reviewed journal. Design: Retrospective, observational study. Subject and Setting: The sample frame included dental clinical trials presented at the conferences of the International Association of Dental Research (IADR), European Orthodontic Society (EOS), European Organisation for Caries Research (ORCA), The American Association of Orthodontists (AAO) and The Australian Society of Orthodontists (ASO) from January 2005 to December 2007. The sample frame for the University Teachers Group (UTG) abstracts, included abstracts presented at the UTG session of the British Orthodontic Conference (BOC) 1999-2010. Sample size Spencer found a publication rate of 38% from abstracts of clinical trials presented at EOS, IADR, ORCA and a 50% increase would be give a publication rate of 57%. Using data from Spencer in Pocock’s formula, 210 abstracts would be required to give 80% power, at the 5% level, and enable me to detect a 50% rise in the proportion of clinical trial abstracts published. Method: Clinical trials presented at above conferences were identified from the associated journals or conference proceedings. Inter-examiner and intra-examiner reliability were assessed using a random 10% sample of abstracts. A MEDLINE search was undertaken to determine whether the abstract had been published in full. The date of publication was recorded. Authors of abstracts that did not reach publication were contacted to determine the reasons. Results: Seven thousand and sixty-nine abstracts presented from 2005-2007 were identified, including 215 clinical trials. 142 abstracts were identified from the UTG session from 1998 – 2008, and all were included. The publication rate for the 2005-2007 sample was 32.6% and the UTG sample 34.5%. There were no predictors of publication in either group studied. The median time to publication of the 2005 – 2007 group was 16.00 months, IQR (10, 26) and the mean time to publication for the UTG group was 18.3 months (95% CI 14.38, 22.19). For the unpublished 2005-2007 group, reasons given for failure to publish were lack of time (8.3%), language, culture, lack of teaching (1.4%), rejection (0.7%), motivation (0.7%), perceived editorial bias (0.7%) and length of review process (0.7%). For the UTG group, reasons given included lack of time (19.4%), lack of interest from SpR (9.7%) or in press (7.5%). Conclusions: No predictors of publication were found for either group studied. For the unpublished 2005-2007 group, main reasons given were lack of time (8.3%), language, culture and lack of teaching (1.4%). For the UTG group, reasons given included lack of time (19.4%), lack of interest from SpR (9.7%) or in press (7.5%). The qualitative results should be viewed with caution due to the low response rate (12.4% for the 2005 – 2007 sample, and 68.8% for UTG).