This thesis has investigated the role of neutrophil extracellular traps (NETs) in the pathogenesis of periodontitis. A fluorometric assay was developed for the in vitro quantification of NET release and used to assess relative levels in chronic periodontitis patients, indicating elevated release under several stimulatory conditions compared with controls. Studies also demonstrated that challenge with periodontitis-associated bacteria evoked differential NET release. In addition, an extensive panel of periodontal pathogens was screened for two potential virulence traits that may confer a survival advantage when challenged with NETs. Deoxyribonuclease (DNase) secretion, capable of degrading NETs, and resistance to killing by histone, a core structural and antimicrobial protein within NETs, were both shown to be relatively common characteristics possessed by pathogens associated with severe disease. Additionally, an in vivo study demonstrated DNase activity in plaque and GCF during the development of experimental gingivitis. Further studies examined the association between neutrophil reactive oxygen species production and NET release implicating a regulatory role for myeloperoxidase generated hypochlorous acid in NET production. In conclusion, the results contained within this thesis indicate a putative role for NETs in the pathogenesis of periodontitis and highlight a high prevalence of potential counter-NET mechanisms in certain periodontal organisms.

Titanium (Ti) and its alloys are considered suitable group of materials to be used for biomedical implants. Ti implant materials are used in both indwelling sites such as parts of joint replacements or in sites that penetrate the epithelium such as dental implants. In both contexts, device failure which is often associated with chronic inflammation is a significant problem. In the current thesis, advanced biophysical imaging techniques employed demonstrated accumulation and speciation of Ti ions and particles in inflamed peri-implant tissues excised from indwelling, percutaneous and transmucosal sites. Ti distributions identified in forms indicative of corrosion processes suggests that indeed Ti should be considered as a potential modifier of the peri-implant physiological processes. Evidently, neutrophils are the predominant acute inflammatory cells in percutaneous and transmucosal peri-implant sites. Therefore, an array of neutrophil immune responses (phagocytosis, respiratory burst, NETosis, cytokine secretion and chemotaxis) were studied following stimulation with Ti in forms identified in the peri-implant tissues. Finally, the modification of neutrophil immune responses to known periodontal pathogens following Ti exposures was explored. The findings strongly indicated that neutrophil responses can be modified by Ti species and further work is needed to identify the role that ‘free Ti’ has on implant outcomes.

Low level light therapy has been widely used in the management of a range of human diseases. Light irradiation triggers a range of cellular signalling processes in a variety of cells, promoting wound healing and preventing cell death. The aim of this study was to investigate the photobiomodulatory effects of low level lasers and light-emitting diodes (LEDs) on human oral epithelial cells (H400 cells) as well as neutrophils, as a potential management strategy for periodontitis. Initially light sources were characterised to obtain dosage (radiant exposure) for light experiments. In addition, a model system utilising H400 cells was developed and characterised prior to laser and LEDs irradiation analysis. Biological responses were determined upon irradiation. Results demonstrated that irradiation by laser and LEDs enhanced H400 cell growth. This was described by mitochondrial metabolic activity and cell proliferation marker, Ki-67. This supports the ability of low-level light to trigger cell growth for further healing inflammation in periodontal disease. Furthermore, ROS production by human neutrophils was attenuated following LEDs irradiation and this suggests this light therapy may decrease level of neutrophil ROS in inflamed tissue and improve wound healing. Data suggested potential therapeutic benefits for enhancing healing in the gingival epithelium, which propose the possibility of the use of light therapy, a non-invasive tool in periodontal disease management.

Although the regenerative capacity of the dentine-pulp complex, induced by pulp capping agents, is well established, the molecular processes by which this occurs, are poorly understood. Calcium silicate cements are powerful stimulators of wound healing in the pulp. This project aimed to investigate the potential of calcium silicate cements to liberate dentine extracellular matrix (dECM) components, characterise growth factors present and determine if these dECM components, or any specific growth factor characterised, had bioactive effects on pulp wound healing. dECM was solubilised from human dentine using solutions of EDTA, calcium hydroxide, white MTA, grey MTA and Biodentine®. Proteomic analysis using cytokine arrays and multiplex ELISA demonstrated the presence of a broad range of growth factors, which were differentially released, suggesting that each agent had differing capacities to liberate such molecules. In vitro analysis of pulp cells exposed to dECM components released by calcium silicate cements, demonstrated increased capacity for cell proliferation and chemotaxis. A previously unidentified growth factor in dECM, hepatocyte growth factor, was shown to induce pulp cell proliferation, migration, differentiation and mineralisation in vitro. Growth factors liberated from dentine by the soluble components of pulp capping agents may regulate reparative events leading to pulp wound healing in vivo.

Mesenchymal stem cells are valuable for regenerative dental research. However, the use of adipose-derived cells (ADCs) within regenerative dentistry remains relatively unexplored. This project aimed to determine an optimal method for the isolation of rat ADCs, to evaluate the influence of cell selection and cryo-storage on MSC phenotype, and compare the relative stemness and dentinogenic capacity of ADCs with bone marrow (BMDCs) and dental pulp-derived cells (DPDCs). Digestion with type-I collagenase for 30 minutes at 37ºC released the greatest number of viable and proliferative ADCs from inguinal adipose tissue. FACS and sqRT-PCR profiling indicated that ADCs shared similar levels of MSC markers (e.g. CD73, CD90, CD105) with BMDCS and DPDCs. The expression of MSC markers was also increased following cryo-storage for all cell types. Alizarin red staining, SEM and micro-CT analyses indicated that the osteogenic differentiation capacity of ADCs appeared lower than that of BMDCs and DPDCs. The FACS procedure reduced cell viability and CD29/CD90 cells had limited osteogenic differentiation capacity when compared to unsorted cell populations. Dentine matrix component (DMCs) supplementation (1 µg/mL) increased the volume of mineralised deposits in ADC, BMDC and DPDC cultures, as well as the expression of odontogenic markers (DMP1 and DSPP) in ADC and BMDC cultures. In conclusion ADCs have an odontogenic capacity, although this may be limited when compared with BMDCs and DPDCs. These findings indicate that when compared with BMDCs or DPDCs, ADCs may have a comparatively limited applicability for dental tissue engineering. However, ADCs can be isolated in comparatively large numbers with relatively little patient discomfort when compared with BMDCs and DPDCs, and these and previous studies have indicated that ADCs can be induced towards a dentinogenic phenotype.

Pocket/sulcular epithelium is the first line of defence to plaque bacteria and its potential role in periodontitis is investigated. This thesis describes the development of a model system, utilising an immortal epithelial cell line (H400) in order to investigate responses to periodontal pathogen stimulation (P. gingivalis and F. nucleatum) in terms of NF-\(\kappa\)B activation, differential gene expression and cytokine production. The pathogenesis of periodontitis suggests that susceptible patients exhibit a hyper-inflammatory response to plaque bacteria, so an attempt to modulate the pro-inflammatory epithelial response using a natural di-thiol antioxidant \(\alpha\)-lipoate was also investigated. Data demonstrated that periodontal pathogens P. gingivalis and F. nucleatum elicited a pro-inflammatory response in the H400 model system. This was confirmed by demonstrating NF-\(\kappa\)B activation, gene expression changes and downstream cytokine production. Ontological grouping of gene expression changes revealed a range of gene functions which support the hypothesis that the epithelium may play a role in the initiation and propagation of the periodontal lesion. In addition, alipoate was able to modulate this inflammatory response but not completely block this essential defence mechanism. Data obtained indicates the potential of utilising \(\alpha\)-lipoate as an adjunct in the management of periodontitis.

Despite recent improvements in the clinical outcomes of vital pulp treatment, existing approaches remain non-specific and unpredictable. Developing biologically-based therapies that promote pulp regeneration is critical. Epigenetic modifications of DNA and histones control cellular processes, including proliferation, mineralisation and stem cell fate, and therefore offer exciting therapeutic opportunities. Chromatin acetylation can be altered pharmacologically using histone-deacetylase-inhibitors (HDACis), which relax its structure and modulate transcription. This project investigated regenerative-associated HDACi effects in vitro on a cell-line and primary dental-pulp-cells (DPCs), using proliferation, viability, mineralisation, cell-migration, enzyme activity, high-throughput gene/protein expression and pathway analyses. HDACis increased DPC differentiation and mineralisation-associated gene/protein expression at concentrations, which did not reduce viability. Primary DPC mineralisation was promoted without altering cell viability/apoptosis, indicating a resistance to HDACi-mediated toxicity compared with cell-lines. HDACi-induced DPC reparative processes were mediated by matrix metalloproteinase (MMP) expression and activity. MMP-13 inhibition further increased mineralisation-associated events, but decreased cell-migration indicating a novel role for MMP-13 in pulpal repair. HDACi solutions released a range of previously characterised and unreported bioactive dentine matrix components, which may further supplement regenerative capability in vivo. Results demonstrate that HDACi directly stimulate DPC repair-associated events, highlighting their potential for augmenting dental materials or pulp-engineering scaffolds for regenerative endodontics.

Inflammation is a central component of the immune response. In its acute form it aids the transition from disease to health via the activation of numerous immune cells, enabling them to reach the site of infection/injury and orchestrate themselves to combat pathogens, facilitating resolution and repair to restore the host to health. However, chronic inflammation is deleterious to the host and differs from the “classical” acute inflammatory process in that the inflammation is not necessarily so readily obvious and is not self-limiting; rather, the immune system is in a constant state of low-grade activation and when challenged by pathogenic or sterile injury the response is heightened, resulting in prolonged tissue damage and a failure of efficient resolution mechanisms. Neutrophils are important mediators of acquired innate immune responses but may also contribute to the pathogenesis of chronic inflammatory diseases. Neutrophils are heavily involved in antimicrobial defence; their primary role is the localisation and elimination of pathogenic microorganisms. This, combined with their relatively short lifespan, has resulted in a traditional view of them as limited “kamikaze” cells. However, as detailed here, neutrophils have been shown to act with complexity and sophistication, orchestrating the immune/inflammatory response but also inadvertently contributing to tissue damage in different disease states. This thesis includes the study of neutrophil function in acute inflammatory episodes such as gingivitis and more chronic long-term health conditions such as obesity, chronic periodontitis and Papillon-Lefèvre Syndrome. The findings outlined here support the role of neutrophils as important contributors to both acute and chronic disease, showing these cells to be far more sophisticated than previously regarded.

The majority of materials used for cemented joint replacement and bone augmentation contain polymethylmethacrylate (PMMA)-based materials. Although commercially available for more than 60 years, PMM-based cements suffer from shortcomings including: the requirement of mixing in surgery of its components, high setting time and formation of a fibrous scar at the bone-implant interface, which may lead to necrosis of surrounding soft and hard tissues and failure of the implant. Incorporation of various fillers has been used to improve the physical, mechanical and biocompatibility with soft and hard tissues of PMMA-based materials, although the formation of a fibrous scar still remains one of the biggest concerns regarding its possibility of integrating with the surrounding bone tissue. Alternative materials have been tested including calcium phosphate cements and bioceramics in an attempt to provide a more suitable material for bone repair compared with PMMA-based cements. This work used a PMMA-based material as a starting point to develop a material with enhanced physical and mechanical characteristics that integrated with surrounding bone without the formation of the fibrous scar at its interface with bone and soft tissues. This new material will be based on the incorporation of different types of bioactive glasses in dimethacrylate-based resins.

This thesis investigated neutrophil extracellular traps (NETs) in the pathogenesis of periodontal diseases, including chronic periodontitis, experimental gingivitis and Papillon Lefèvre syndrome (PLS). \(In\) \(vitro\) assays investigated the interactions between periodontal bacteria and peripheral neutrophils isolated by discontinuous Percoll gradients, and demonstrated differential NET release in response to bacteria. Interestingly, NETs entrapped all periodontal bacteria assayed to some extent; however bacterial growth and survival were not impeded. A longitudinal intervention clinical study of chronic periodontitis patients and matched healthy controls revealed no differences in peripheral NET production; however NET production by patients decreased following non-surgical treatment. Furthermore, a subset of patients displayed impeded NET degradation by plasma that was restored following disease treatment; this may be the result of increased circulating immunoglobulins and free light chains (FLCs) pre-treatment. Peripheral NET production did not change throughout the experimental gingivitis model study; however NET release was impeded in PLS patients relative to healthy controls. Additional in vitro studies demonstrated that cigarette smoking had an inhibitory effect on NET release. Collectively, this thesis indicates that NETs contribute to innate immunity, however, given that periodontitis pathogenesis is characterised by aberrant neutrophil responses, NETs may also be involved in the progression of the disease.