Dental pulp progenitor cells (DPPCs) are among many stem cell sources potentially beneficial for tissue engineering. DPPCs offer advantages over other mesenchymal stem cell sources, due to their accessibility and multi-lineage differentiation. However, distinct DPPC clones exist within dental pulp, with contrasting proliferative/regenerative capabilities. This is a key consideration for the exploitation of DPPCs, in terms of the abilities of isolated clones to undergo sufficient in vitro proliferative expansion, while maintaining their regenerative potential. This Thesis supports the heterogeneous nature of DPPCs, demonstrating significant variations in population doublings (PDs) and senescence, with highly proliferative clones exhibiting greater proliferation (>80PDs) under normal and oxidative stress (H2O2 treatment) conditions, compared to low proliferative clones (<40PDs) demonstrating altered morphology, increased SA-β-galactosidase staining and senescence marker (p53/p16) expression. Although negative for human telomerase expression, highly proliferative clones possessed longer telomeres (>18kb); maintained stem cell marker expression (CD73, CD90, CD105) and osteogenic/chondrogenic differentiation in culture. In contrast, low proliferative clones exhibited shorter telomeres (<6kb), reduced marker expression and increased adipogenesis. DPPC heterogeneity was further evident upon Raman Spectroscopy analysis, which distinguished between undifferentiated high and low proliferative clones and undifferentiated DPPCs and clones following chondrogenic differentiation. High and low proliferative clonal behaviour was also assessed in type I collagen gels, demonstrating increased contraction and reduced cell proliferation in detached versus attached gels, irrespective of clonal type. Increased contraction was due to increased MMP-2 expression by both clonal types, while highly proliferative clones also expressed MMP-9, especially at late PDs. These findings indicate significant variations in DPPC clonal proliferative/differentiation capabilities, partly explained by telomere length/cellular ageing differences. Identification of clonal populations with contrasting tissue regeneration capacities supports the development of screening strategies (e.g. Raman Spectroscopy) for the selective isolation of highly proliferative clones from dental pulp for therapeutic use, or assessing DPPC differentiation in 3D culture.

Periodontitis is a widely spread disease, affecting about 80% of the worldwide population, resulting in teeth loss, a heavy impact on patients in terms of function and aesthetic. Type 2 Diabetes Mellitus (T2DM) is described to be linked to the exacerbation of periodontitis and delayed healing. This link between these two diseases, however, is not fully evaluated and the mechanisms are yet to be fully elucidated. Osteopontin (OPN) is described to inhibit mineral crystal formation. Herein, it has been hypothesized that increased OPN in diabetic healing bone may be the causative factor of delayed healing in periodontitis and subsequent deterioration, leading to teeth loss. This project aims to gain a greater understanding of the effect of high glucose (HG) levels on mesenchymal stem cells (MSCs) and macrophages, their ability to synthesise OPN and hence, its effects on MSCs to synthesise new bone tissue. Further, the influence of Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) on these cells was analyzed, in an attempt to create a model to study the healing in the presence of periodontitis and T2DM. Investigating the MSCs isolated from the rat compact bone (CB-MSCs) during the growth in culture, revealed two main populations; a heterogenous population appeared with predominantly mature characteristics at PD15. This population then demonstrated a change in its heterogeneity and became more immature in nature at PD50. These two main populations differed in their growth rate and capability of osteodifferentiation. HG environments exerted significant decreases in osteogenic differentiation on PD15, but not PD50. Addition of pg-LPS showed inhibitory effects on osteodifferentiation on PD15 cells more than PD50. Conversely, in the combined presence of HG and pg-LPS, PD50 showed a significant decrease in osteodifferentiation. OPN levels demonstrated a gradual decrease in CB-MSCs in both normal and HG conditions. Investigating OPN levels secreted by macrophages, however, revealed interesting results. Synergistic effects of both HG and pg-LPS exhibited a significant increase in OPN levels in both pro-inflammatory M1 macrophages and in repair related M2 macrophages. In conclusion, HG was mainly reported to inhibit osteogenic differentiation of the mature cell population, whereas the immature population was found to be affected by combined pg-LPS and HG. OPN levels in HG conditions were shown to decrease along the osteodifferentiation period. However, macrophages showed increase secretion of OPN by the synergistic effects of both pg-LPS and HG in both M1 and M2 and by pg-LPS effects in M2 macrophages. These outcomes as far as we are aware, are novel and disclose a new mechanism of bone resorption in the case of T2DM patients concurrently with periodontal disease.

Denture-associated stomatitis (DS), a frequent infection in denture-wearers (up to 60%), presents as areas of palatal inflammation and is normally associated with denture biofilms containing Candida albicans. However, the contribution of co-existing bacteria in these biofilms to the infection remains unclear. As current DS management strategies are primarily directed towards Candida, research demonstrating the impact of specific bacteria upon infection prognosis is important to improve treatment regimes. This research evaluated the in vitro impact of bacteria on Candida virulence, and compared bacterial microbiomes at specific oral sites in DS and non-DS patients to determine associations with infection. In vitro biofilm studies assessed expression of C. albicans virulence factors (morphological transformation, adhesins, hydrolytic enzymes) and their impact on pathogenesis in an infection model. In clinical studies, microbiological samples were obtained from the tongue, palate and denture-fitting surface of 19 denture-wearing patients (DS n=8, non-DS n=11). The presence of Candida was ascertained by PCR. Bacterial DNA was extracted and subjected to next generation sequencing using bacterial 16S rRNA gene targets, and differences in the bacterial microbiomes determined. Certain bacterial species in acrylic biofilms significantly (P < 0.05) increased the expression of C. albicans virulence factors, and subsequently, enhanced tissue damage in model systems. Candida was detected in clinical samples of 14 patients (DS n=6, non-DS n=8). Metataxonomic analyses revealed differences in relative abundance of bacterial species, but no significant differences in the bacterial microbiomes of the denture-fitting surface and palate between DS and non-DS patients. Importantly, a significant (P=0.007) increase in the number of bacterial species was evident for the tongue microbiome of non-DS patients. The in vitro modulating capacity of bacteria toward Candida virulence, and the observed species-level differences in bacteria between DS and non-DS patients highlight the need for consideration of the bacterial composition of oral biofilms in the pathogenesis of DS.

Candida infections of humans are an increasingly prevalent problem. Most candidal infections are superficial, occurring on mucosal surfaces, however, systemic infection can arise in immunocompromised individuals and these are life threatening. Candida is recognised by host immune cells through pattern recognition receptors e.g. dectin-1. The immune cells then produce cytokines to drive adaptive immune responses. The type of T-helper (Th) cell response that occurs is an important factor in whether candidal colonisation or clearance occurs. The overall focus of this research was to employ in vivo and in vitro studies to assess the nature of the immune response to Candida albicans. Increased pro-inflammatory Th17 and Th1 responses were evident in denture stomatitis (DS) patients based on cytokine profiles. In chronic hyperplastic candidosis (CHC) tissues significantly higher levels of CD4+, IL-12A+, IL-17A+ and EBi3+ positive cells were detected by immunohistochemistry (IHC) compared to control tissues. This finding was indicative of an increased Th17 response in CHC. IHC detection of individual cytokine subunits in cells was more difficult to interpret, but suggested both IL-35 and IL-12 cytokines were present, indicating Treg and Th1 cell responses, respectively. Challenge of a human monocyte cell line and human peripheral blood mononuclear cells (PBMCs) with C. albicans resulted in IL-23 cytokine expression indicative of a Th17 response. Interestingly, lipopolysaccharide (LPS) was important in enhancing the ability of dendritic cells to recognise and phagocytose C. albicans via dectin-1. The recall response from PBMCs stimulated with C. albicans resulted in a significant Th17 recall response. Extrapolation of these findings to the host interaction with Candida requires additional clinical studies and assessment of other forms of superficial mucosal candidosis. This research does however indicate that host recognition of C. albicans leads to a predominantly pro-inflammatory Th17 response.

Recurrence and persistence of microbial infection is one of the main reasons for the revision of dental restorations in the clinic. Failure to control the pathogenic microbiota leads to the formation of caries, which in turn may lead to irreversible pulpal damage,resulting in the need for root canal (endodontic) therapy. Secondary endodontic infections can spread to the surrounding oral tissues and beyond, leaving the patient vulnerable to systemic infection. This project aimed to develop a novel, injectable hydrogel containing antimicrobial liposomes for use as an intracanal medicament in order to reduce the incidence of secondary endodontic infections. Triclosan, a broad-spectrum, hydrophobic antimicrobial drug, was shown to have a bacteriostatic and bactericidal effect against two oral pathogens, Enterococcus faecalis and Streptococcus anginosus, which were grown planktonically and as a single-species biofilm. The bacteriostatic effect was also seen when triclosan was encapsulated in multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs) of phosphatidylcholine:cholesterol (PC:C) liposomes. Antimicrobial efficacy was associated with a high drug:lipid ratio in the liposomes. The liposomes were incorporated into a methyl cellulose (MC) solution, and the rheological properties were measured. MC was a sheer-thinning, viscous solution at ambient temperature and formed a hydrogel as the temperature was increased above 30 C. These properties were unaffected by the addition of liposomal MLVs or SUVs. The hydrogels containing triclosan-loaded liposomes had an antimicrobial effect when incubated in contact with suspensions of E. faecalis or S. anginosus, but MC containing triclosan only did not. A release assay showed the release of triclosan from MC loaded with triclosan liposomes, which was not seen when triclosan was incorporated into MC alone. The liposomal hydrogel was injected into endodontically prepared extracted human teeth that had been inoculated with E. faecalis suspension to mimic endodontic infection. After 24 h treatment, histological analysis showed that triclosan solution,triclosan liposomes and triclosan liposomes in MC prevented the formation of a biofilm on the intraroot surface, which was observed in controls that underwent no treatment or treatment with MC hydrogel only. The results of this work indicated that triclosan liposomes have potential to prevent secondary endodontic infections and may be loaded into a hydrogel suitable for injection into the root canal and subsequent gelation upon thermoequilibriation with the oral cavity. Triclosan release from this hydrogel may facilitate the prevention of bacterial colonisation of the root canal by E. faecalis, which has high prevalance in secondary endodontic infections.

Bacterial and fungal biofilms are an increasing clinical challenge, from non-healing wounds to chronic lung infections in cystic fibrosis (CF) patients. Escalating antimicrobial resistance has led to a need for alternative treatments. OligoG (a low MW alginate oligosaccharide), can disrupt multi-drug resistant bacterial biofilms and decrease antibiotic resistance. This study characterised the interaction between OligoG and the most prevalent CF pathogen, Pseudomonas aeruginosa, using nanoscale characterisation, imaging and fluorescent conjugation. Further investigation into the effect of OligoG on CF sputum was carried out using Fourier transform infrared spectroscopy (FTIR) and rheology. The work was extended to observe changes in fungal pathogens treated with OligoG. Electrophoretic light scattering (ELS) and dynamic light scattering revealed that the surface charge of P. aeruginosa became more negative when treated with OligoG (P<0.001) with an increase in sizing. These interactions were not disrupted by hydrodynamic shear (P<0.0001). Biofilm inhibition and disruption of a mucoid P. aeruginosa strain, treated with OligoG, was demonstrated using confocal laser scanning microscopy (P<0.05). Fluorescent conjugation to OligoG revealed its distribution throughout the biofilm. In vitro scanning electron microscopy (SEM), atomic force microscopy (AFM) and ELS of mucin showed disruption in aggregation when treated with OligoG (P<0.005), with the surface charge becoming more negative (P<0.0001). Ex vivo treatment of CF sputum with OligoG analysed using FTIR and rheology, demonstrated possible interaction with the sulfate moiety of mucin and a reduction in the viscous and elastic response (0.16 Hz; P<0.0001). AFM and SEM analysis of candidal biofilms treated with OligoG demonstrated a dose response in reducing biofilm formation, with a decrease in hyphal formation. An in vitro epithelial model demonstrated these changes at <2% OligoG. These studies provide insight into the role of OligoG as a treatment for CF patients. Furthermore, promising results have shown that OligoG may lower candidal pathogenicity.

Spinal cord regeneration following injury represents a major clinical challenge. Over recent years, stem cells have demonstrated promise for promoting spinal repair in the lab and in early stage clinical trials through functional replacement of neuronal and glial cells and through secondary trophic mechanisms to promote endogenous regeneration. Research has primarily focused on the use of embryonic tissue-derived stem cells of potentially limited therapeutic application due to related ethical concerns. The dental pulp harbours a source of easily accessible progenitor cells that have demonstrated early promise in improving functional outcome of experimental models of spinal cord injury via growth factor release. This thesis explores the potential of dental pulp progenitor cells (DPPCs) to promote spinal repair through direct cellular replacement. Progenitor cells isolated from murine incisors were found to express early stage neural and glial markers. Specific protocols were developed demonstrating the ability of DPPCs to differentiate in vitro into neuronal-like and oligodendrocyte-like cells with appropriate morphology and expression of mature markers. Electrophysiological testing revealed that DPPC-derived neuronal-like cells were of an immature non-functional phenotype. Undifferentiated DPPCs injected into an ex vivo spinal cord slice model showed signs of proliferation, migration and spontaneous differentiation within spinal tissue. DPPCs pre-differentiated into oligodendrocyte-like cells failed to survive transplantation but neuronally pre-differentiated cells survived, showing signs of integration into endogenous neuronal pathways. In a small scale pilot study, neuronally pre-differentiated DPPCs were transplanted into a clinically relevant in vivo model of spinal cord injury. DPPCs maintained expression of neuronal markers four weeks after grafting into the injured spinal cord. Axonal projections towards grafted cells and synaptic protein expression suggested possible integration into neuronal pathways, albeit without an associated statistical functional improvement. The results presented in this thesis provide a strong case for the potential of DPPCs to facilitate functional recovery through direct cell replacement mechanisms.

The main goal of bone repair is to regenerate pre-existing properties and restore tissue integrity and function. It has been reported that bone contains numerous growth factors which are proposed to be released from the matrix during injury and mediate the repair process. These molecules act in synergistic action causing recruitment of progenitor stem cells to sites of bone injury, which then proliferate and differentiate into mature bone synthesising cells capable to initiate repair processes. It has been demonstrated that combinations of growth factors, such as the combinations found in the bone matrix, may be more effective in promoting bone healing compared with single growth factor therapy. This project focuses on understanding bone repair processes by stimulating ex vivo fractured rat mandible model with either endogenous growth factors released by chemical treatment or by exogenous single growth factor therapy and investigating their effects on cellular behaviour. This project utilised the ex vivo mandible model as a promising alternative to current model and fractures were made within the ex vivo mandible slices to mimic bone fracture repair scenario. In summary, ex vivo experimental models were used successfully to investigate mechanism of bone repair. The results demonstrated that bioactive growth factors, particularly TGF-β1, BMP2 and VEGF successfully released from the bone matrix by EDTA, citric acid and calcium hydroxide. These growth factors found to affect cellular behaviour, by influencing proliferation and differentiation of osteoprogenitor cells. Calcium hydroxide derived endogenous growth factors mediated the repair process of mandibular bone greater than exogenously applied BMP2. Calcium hydroxide may provide a novel therapeutic approaches to utilise the synergistic effect of cocktail growth factors entrapped in bone matrix to stimulate optimal bone regeneration and avoid issues regard single growth factor therapy.

Despite the rich oral microflora, infections within the oral cavity are rare. Rapid wound healing within the oral mucosa occurs, potentially due to the presence of oral mucosa lamina propria-progenitor cells (OMLP-PCs). OMLP-PCs are a novel population of multipotent cells known to possess immunosuppressive properties,through contact-independent mediated mechanisms. Many immunomodulatory soluble factors are also documented to have dual functions as antimicrobials; leading to the hypothesis that OMLP-PCs possess antibacterial properties in addition to their published immunoregulatory actions. The aim of this study was to investigate the antibacterial properties of OMLP-PCs and to define the mechanisms of action. A further aim of this study was to determine whether the antibacterial potential of OMLP-PCs was affected during disease, specifically Graft Versus Host Disease (GVHD). The antibacterial properties of OMLP-PCs were compared between cells isolated from healthy donors and patients with oral chronic GVHD. During this study it was determined that OMLP-PCs possess constitutive antibacterial properties against Gram positive and Gram negative bacteria which are mediated through the release of soluble factors. LL37 and Indoleamine 2,3-Dioxygenase are known to mediated the antibacterial properties of bone marrow-mesenchymal stem cells, however this study determined that these factors did not play a role in the OMLP-PCs antibacterial effects. It was established that osteoprotegerin, haptoglobin and prostaglandin E2 in part mediate the antibacterial effects of OMLP-PCs. For the first time, direct antibacterial properties of osteoprotegerin were demonstrated against Gram positive bacteria. Furthermore, OMLP-PCs isolated from GVHD patients did not display antibacterial properties. It was further established that the secretion of innate cell chemoattractants was dysregulated in OMLP-PCs isolated from GVHD patients compared to healthy controls. This finding demonstrates that during GVHD, the oral mucosa is unable to regulate the oral microflora and sufficiently recruit innate immune cells during infection.