There is little definitive information about the outcome of guided tissue regeneration (GTR) and access flap surgery (AF) in aggressive periodontitis (AgP) patients. This study is the first randomised controlled trial to assess the clinical and radiographic outcomes of both treatments, using 18 AgP patients. The results showed that both therapies were effective in the treatment of intrabony defects in AgP patients, although no significant differences between them could be demonstrated. To determine whether changes in the production of specific wound healing mediators are associated with the successful outcome of periodontal surgery, gingival crevicular fluid (GCF) was collected from affected (GTR and AF sites) and healthy non-affected control areas at various times post-surgery and the components analysed. In contrast to previous studies using conventional enzyme-linked immunosorbent assay (ELISA) which measures only one mediator, the present study developed, validated and then successfully utilised a multiplex-bead-assay (MBA) to simultaneously measure multiple growth factors in small volumes of GCF. These included angiopoietin-1 (Ang-1), vascular-endothelial growth-factor (VEGF), bone morphogenetic protein-2 (BMP-2), osteoprotegerin (OPG), tissue-inhibitor of matrix metalloproteinase-1 (TIMP-1), basicfibroblast growth-factor (bFGF), keratinocyte growth-factor (KGF) and platelet-derived growth-factor-AB (PDGF-AB). GCF volumes were found to significantly increase at all sites 3-7 days post-surgery but thereafter declined to baseline levels except for the GTR sites, which remained elevated even at 42±3 days. Increased amounts of the 8 mediators were observed in the GCF of all groups compared with the baseline pre-surgery, with the GTR group having significantly more Ang-1, OPG and TIMP-1 at 3-5 days and BMP-2 at 7±1 days postsurgery. The GCF at the GTR sites also contained significantly greater amounts of Ang-1, VEGF, BMP-2 and OPG compared with the unaffected control sites, but there were no significant differences in the amounts of any of the mediators between the GTR and AF groups. Notably, the amount of KGF and the concentration of PDGF-AB in GCF were found to be significantly associated with a successful clinical outcome at both 6 and 12 months following GTR surgery. In conclusion, although the clinical outcomes did not differ significantly between the 2 surgical treatments of the AgP patients, each therapy was found to differentially affect the profiles of the mediators in GCF post-operatively. Moreover, the new MBA which was developed here was capable of successfully measuring multiple components in small volumes of GCF, indicating that this technique might also be of value in assessing the specific molecular changes in other healing wound fluids, and thus possibly a useful prognostic indicator of the outcome of clinical intervention.

Background: Amelogenesis Imperfecta (AI) is an inherited dental condition affecting enamel, which can result in significant tooth discolouration and enamel breakdown, requiring lifelong dental care. The possible impact of this condition on children and young adults is not known. Aims and Objectives: The aim of the study was to explore the impact of AI on children and young adults through in-depth interviewing and subsequent Framework Analysis. The information derived from this was then used to construct a questionnaire. Methods: This research comprised of two parts, combining qualitative and quantitative methodology, in order to develop a questionnaire to distribute to a large cohort of AI patients. The first part involved semi-structured in-depth interviews with 7 AI patients and common themes and concepts were then identified using Framework Analysis. The second part of the study was the development of a questionnaire based on the themes and subthemes identified from part one of the research. This questionnaire was then distributed to 61 AI patients mixed between three cohorts of AI patients: pre, mid, and post-treatment. Results: Children and adolescents with AI exhibited concerns regarding the aesthetics and function of their dentition. Patients also expressed a high level of concern regarding comments by other people and self consciousness associated with this. A small number of AI patients highlighted the effect of their dental treatment and health on their personal life. Conclusion: The results indicate that there are marked impacts on children and young adults as a result of AI. These include aesthetics, function, and psycho-social aspects.

Infected wounds are a major cause of hospital-acquired infections and these are difficult to treat due to the emergence of antibiotic-resistant bacteria. This project is concerned with evaluating a novel antimicrobial approach involving the photosensitizer indocyanine green (ICG) which generates reactive oxygen species when irradiated with near-infrared (NIR) light which enables good tissue penetration. The photo-susceptibility of common wound-infecting organisms to ICG coupled with NIR-light was investigated. All species were susceptible to killing. ICG at a concentration of 25 μg/mL enabled the killing of the Gram-positive species (Staphylococcus aureus and Streptococcus pyogenes), higher concentrations (100-200μg/mL) were necessary to achieve substantial kills of the Gram-negative species (Pseudomonas aeruginosa and Escherichia coli). Both high and low fluences were able to kill 99.999% of the Gram-positive bacteria. High fluence irradiation was necessary to kill 99.99% of the Gram-negative bacteria. The pulsed-mode of irradiation was as effective as the continuous-mode for killing the Gram-positive species. Yet only the continuous-mode of irradiation was able to kill P. aeruginosa. Biofilms of Staph. aureus and P. aeruginosa were susceptible to disruption and killing by ICG-photosensitization. A significant enhancement of lethal photosensitization of Staph. aureus was achievable using gold-nanoparticles and antioxidants. Significant kills (>99%) were achieved in the presence of serum and 100 μg/mL ICG. A low oxygen concentration reduced the kills to 96.77% and 71.62% for Staph. aureus and Strep. pyogenes respectively. Mechanistic studies revealed that killing was mediated mainly by reactive-oxygen species. In vivo studies in mice showed that ICG and continuous-NIR light could achieve kills of 96%, 93% and 78-91% for P. aeruginosa, Strep. pyogenes and Staph. aureus respectively. The results of these in vitro and in vivo studies imply that ICG-PDT could be an effective means of decreasing the microbial burden in wounds.

Introduction: Methods to determine a child’s growth and development are of a great value for both medical and dental practice. Growth has been used as an indicator of health worldwide. Normal development is measured from growth charts and any developmental delay can be recognized early. Teeth are the most frequently used part of the body analyzed for age estimation. Aim & Objectives: 1. To revise the dental development timings for permanent teeth (excluding third molars) among the UK population using dental radiographs and produce reference data for different ethnic groups. 2. A pilot study to revise dental development timing for primary teeth using post mortem study of MRI and CT scan (MaRIAS) data. Methods: Cross sectional study of radiographs of children attending Eastman Dental Hospital and MaRIAS images (MRI and CT Scan) of fetuses, infants and children at Great Ormond Street Hospital. Tooth development stages (TDS) were assessed using Demirjian’s staging system for permanent teeth and the modified Demirjian’s staging system for primary teeth, to produce mean ages of attainment for tooth initiation, crown completion and root completion. The mean age of attainment of each TDS was produced. Gender and different ethnic groups were compared where appropriate. Results: 1. In total, 183 cases from post-mortem study of MRI and CT scan were assessed (MaRIAS). Of these, 81 cases showed primary teeth, of which 45 (56%) were included in the analysis. Mean ages of attainment of TDS in primary teeth were produced. 2. Three hundred seventy six (376) radiographs of young children and adolescents and 34 MaRIAS images, were assessed to obtain data on the early developing permanent teeth, as this information was lacking from the DAA database. The mean age of attainment of TDS for upper and lower left teeth were produced. 3. Of 5333 subjects in DAA database, 4397 were analysed to compare the mean age of attainment between ethnic groups. Gender considered separately. Conclusion: The primary teeth started to develop in intra-uterine phase. In permanent teeth, females developed earlier than males and there were significant differences when White, Asian and Black groups were compared.

Muscle-derived cells (MDCs) are a heterogeneous population consisting of cells that can undergo myogenic differentiation; however, it has emerged that not all MDCs are restricted to the myogenic lineage. This discovery may have many implications; for example, MDCs may be a suitable alternative source of osteogenic cells for bone repair. The currently accepted treatment for bone repair, bone grafting, is often associated with small amount of obtainable bone. Much of the work published regarding the differential potential of MDCs has not, to date, focused on the osteogenic pathway and even fewer studies have been performed on human cells. In this thesis osteogenic MDCs were isolated by differential adhesion to fibronectin (Fn) i.e. MDCsFn and compared with mesenchymal stem cells (MSCs) in relation to their osteogenic potential. The osteogenic potential was assessed by measuring mineralization and relevant gene- and protein- expression. MSCs and MDCsFn had a similar pattern of ALP activity and expression. Furthermore, MSCs and MDCsFn both showed mineralization after 3 weeks measured by Alizarin Red S. A qPCR Array measuring the activity of 46 osteogenic genes also showed similarities in gene expression between the two cell types; however, the MSCs showed a more consistent pattern between patients, compared to MDCsFn. Titanium (Ti) has previously been used as a bone repair scaffold in humans due to its osteoconductivity. The interaction between Ti, of various roughness and hydrophilicity, and the two cell types, i.e. MSCs and MDCsFn, were assessed with relation to biocompatibility. Interestingly, the hydrophilic, rough surface, which has been described as superior in bone formation applications, showed higher levels of cell death, both apoptosis and necrosis, compared to the other tested surfaces for both cell types. In conclusion, due to the similarities between MDCsFn and MSCs there might be possibilities to use the osteogenic fraction in future bone regenerative applications.

Hepatitis C virus (HCV) is an increasingly common cause of liver disease worldwide. HCV may be present in saliva suggesting possible transmission via oral fluids. However the precise influence of peripheral blood carriage of HCV upon the frequency and load of HCV in oral fluids remains unknown. The objectives of the study were to determine the frequency of oral carriage of HCV and determine the influence of plasma and peripheral blood mononuclear cells (PBMCs) HCV levels upon HCV levels in oral fluids. The study group comprised 85 patients with treated (18) or untreated (67) HCV infection resident in northern Brazil. HCV 5’-NCR and NS5b regions were detected by quantitative Real Time PCR and nested block based PCR respectively in whole saliva, plasma, CD2+, CD14+, CD19+, and CD45+ PBMCs for both methods. Approximately 32% of the total group had detectable HCV RNA in whole saliva. In contrast HCV was present in 82.5% of plasma and up to 82.1% of PBMC. There was no significant difference in the genotypes between oral and blood compartments and no evidence of genetic diversity within the oral compartment. There was no correlation between the salivary prevalence of HCV with load of plasma. HCV RNA viral load in saliva was almost always below the level of quantification. Given the low viral load presented in patient with a high plasma titration in the 30% saliva positive patients, HCV transmission via the saliva is highly unlikely, but cannot be discarded. There is no evidence for HCV compartmentalization as sequences from different compartments were closely related, although mutations were identified more frequently within genotype 1b in all compartments. This finding suggest a new trend towards hepatitis C evolution, as genotypes 1a and 1b do not usually differentiate in terms of treatment outcome, being both traditionally related to poor antiviral response.

Pre-clinical safety testing (in vivo and in vitro) of the therapeutic monoclonal antibody (mAb) TGN1412 (developed for the treatment of autoimmune diseases) failed to predict the life threatening adverse events that occurred during its Phase I Clinical Trial. The treatment of disease using mAb therapy is becoming increasingly common, so, to ensure the safety of mAbs, pre-clinical safety tests that can better predict the toxicity of immunomodulatory mAbs, such as TGN1412, are required. The aim of this study was to investigate the hypothesis that cytokine-driven adverse effects of therapeutic monoclonal antibodies and the mechanisms involved can be better predicted with novel in vitro procedures using human cells, given the failure of animal models to predict the toxicity of TGN1412. Consistent with the results from pre-clinical testing, aqueous phase TGN1412 incubated with human peripheral blood mononuclear cells (PBMC) failed to stimulate the “cytokine storm” suffered by the six recipients of TGN1412. In contrast, TGN1412 immobilised onto polypropylene microtitre plates by “air-drying” stimulated cytokine release from PBMC. This technique was superior to other mAb immobilisation techniques, investigated in terms of predicting cytokine release. Immobilisation of TGN1412 may mimic the immunological synapse formed between this mAb and target cells in vivo. In a more physiologically relevant procedure, TGN1412 incubated in aqueous phase with PBMC over a monolayer of human endothelial cells stimulated cytokine release. Endothelial cell to PBMC contact was crucial to these responses. Furthermore, interactions between lymphocyte function-associated antigen-3 (LFA-3) and intercellular adhesion molecule-1 (ICAM-1) expressed by endothelial cells with their counterstructures CD2 and LFA-1, respectively, expressed by T cells, mediated these TGN1412-stimulated responses. Both procedures developed in this study were capable of distinguishing therapeutic mAbs not associated with a significant incidence of cytokine-driven clinical infusion reactions from mAbs frequently associated with clinical infusion reactions.

Absent or defective craniofacial skeletal muscle can lead to loss of function and aesthetics. Current therapies are fraught with limitations: for example, the surgical transfer of tissue is associated with donor site morbidity coupled with a paucity of available tissue. The potential to engineer skeletal muscle tissue could circumvent disadvantages related to current techniques. Furthermore, the creation of a muscle testbed could provide the means to investigate the response to various manipulations. The aim of this research was to produce an in vitro human craniofacial skeletal muscle tissue suitable as a test-bed for novel therapies involving the craniofacial region. Degradable phosphate-based glass fibre scaffolds of various configurations, combined with extracellular matrix (ECM) components, were seeded with human craniofacial muscle-derived cell cultures. Myogenicity was confirmed with immunofluorescent techniques prior to seeding. The seeded scaffolds were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for up to 21 days. Modulation contrast microscopy was used to analyse migration and morphology. Cell attachment and survival were assessed with the CyQUANT® and alamarBlue® assays, and cell differentiation and maturation were investigated using immunofluorescence and quantitative RT-PCR. Parallel arrays of glass fibres coated with ECM components provided the correct topology to support cell alignment and differentiation. Specifically, compared to control scaffolds, glass fibre scaffolds promoted upregulation of developmental, fast and slow myosin heavy chain genes. Further refinement of the system involved glass fibres embedded within collagen gels, created to mimic the architecture of native skeletal muscle: cells within these constructs were aligned parallel to the glass fibres, and over time, the constructs rolled along the short axis to produce a muscle ‘organoid’. Additionally, the collagen gel contracted along the long axis to reveal tufts of glass fibres analogous to tendons (mean 13.87% reduction in length). Upregulation of the myosin heavy chain genes was promoted, albeit at a later timepoint. In conclusion, degradable glass fibre-ECM scaffolds provided the correct topographical and biological cues to aid the in vitro engineering of human craniofacial skeletal muscle tissue.

This study is aimed towards the appreciation of the structure-activity relationships of polymeric materials with regard to their capability to form coatings that inhibit common oral diseases, and addresses the need for new actives and technologies for improved dental care. Potential tooth-coating materials, including a series of poly(alkyl methacrylate)s, have been selected and evaluated for their capacity to resist acid demineralisation, to inhibit staining and to prevent the pain associated with dentine hypersensitivity. Systematic variation of the length of the pendent alkyl chain (from ethyl to octadecyl) has shown that a poly(butyl methacrylate) forms films of good quality on dentally relevant substrates. The data have indicated that the glass transition temperature of the material is the single most important factor determining film quality. To facilitate formulation in dental care products, miniemulsion methods have been used to suspend this polymer into an aqueous medium as highly stable nanoparticles with an average diameter of less than 100 nm. The integrity of the coatings prepared from these nanoparticles has been assessed using microscopic imaging techniques and has been tested in challenges designed to mimic those imposed by the oral environment. Poly(butyl methacrylate) films have been shown to provide a substantive coating that inhibits acid demineralisation and staining, and prevents dentine hypersensitivity.

Whilst considerable temporal microbial stability has been demonstrated within the oral cavity, the mechanisms responsible for the development, maintenance and stability of individualspecific human oral microbiotas are not fully understood. This doctoral thesis presents a series of in-vitro and ex-vivo investigations that focused on four factors believed to be involved; the microbial factors i) colonisation resistance (CR; a consortial-intrinsic homeostatic mechanism); ii) probiosis (the introduction of bacteria claimed to positively influence the microbial balance of human microbiotas); and the host-derived factors iii) antimicrobial peptides (innate immunity); and iv) secretory antibodies (homoral immunity). To investigate microbial homeostasis in the absence of immunological influences (i.e CR), long-term multiple sorbarod oral microcosms were established using fresh saliva from three volunteers (designated A, B and C). Following the establishment of dynamic steady-states, a reciprocal exchange of pcrfusates (analogous to dispersed plaque bacteria) was performed between MSDs A and B and the resistance of microbial profiles to immigration was tested by differential culture and PCR-DGGE. Fermenter C was maintained to investigate long-term stability of modelled plaque in the absence of host immune factors. 1fSD-C maintained short-term dynamic bacterial stability but streptococci and lactobacilli decreased to undetectable levels after 20d. Whilst increases in streptococci and reductions in lactobacilli occurred in MSDs A and B following mixing, CR was apparent as both communities maint.ained their original DGGE-profiles. CR together with transient stability in unmixed in-vitro plaques indicates the importance of intrinsic homeostatic mechanisms in maintaining short-term stability but that additional factors must be responsible for long-term stability in-vivo, such as the innate and adaptive immune systems and bacterial immigration. The potential influence of the oral probiotic bacterium Lactobacillus reuteri was studied in constant depth film ferrnenter plaques. Dosing in-vitro, plaques with L. reuteri formulations resulted in decreases in Gram-negative anaerobes (6 to 4.5 10glOCFU/mm2) and significant decreases in streptococci (7 to 4 loglOCFU/mm2). Morphological and q-PCR tracking of the probiotic indicated its persistence three weeks after treatment was ceased. Addition of L. reuteri to invitro oral microbiotas therefore markedly influenced microbial composition reaffirming the role of external factors in maintenance of homeostasis. The effect of host defence peptides (HDPs; innate immune factors) on maintenance of stability of in-vitro plaques was therefore tested. The peptides (defensins, histatins and a cathelicidin) caused differential decreases in bacterial viability and the ~ defensins and His-5 additionally inhibited coaggregation. Peptide-exposed plaques exhibited closest similarity to salivary incocula following exposure to the peptides in combination, according to DGGE analyses. These observations indicate a role for physiological levels of host defence peptides in influencing the composition of oral microbiotas. In order to investigate the role of salivary immunoglobulins (adaptive immunity), saliva from three volunteers was fract.ionated by centrifugation into antibody (supernatant) and bacterial (pellet) fractions and streptavidin-coated magnetic bead separation was used to identify and retain bacteria according to their recognition by host-specific Igs. Bead-selected bacteria were then profiled using eubacterial PCR-DGGE and cluster analyses and identified by DNA sequencing. Analyses indicated that whilst a large proportion of oral bacteria (c. 50% of DGGE bands) were recognised by salivary IgA and IgG, the specificity of host antibodies for exogenous oral bacteria and putatively transient species was great.er in all cases. These investigations indicate that the composition and stability of oral microbiotas are variously influenced (individually and probably combinatorially) by the adaptive and innate immune systems, by CR and by bacterial immigration (as occurs naturally and as applied in probiosis).