Objectives: This research aimed to characterize the interfacial characteristics of modified dental resin-based adhesive systems bonded to sound and carious dental tissue. The modification included the incorporation of matrix metalloproteinase (MMP) inhibitors within the primers of these adhesives. Materials and methods: Two MMP inhibitors (BB94 and GM6001) were added to three adhesive primers, Optibond FL “OB” (Kerr, USA), Prime&Bond NT “PB” (Dentsply, USA) and G-Bond “GB” (GC, UK) and bonded to sound dentine. The inhibitory effect of the modified adhesive on recombinant MMPs and on sound dentine MMPs was assessed using FRET-based measurement of MMP activity and substrate zymography, respectively. Micro-tensile bond strength and micro-permeability were used to evaluate the modified adhesives’ physical properties. Micro-Raman spectroscopy analysis was validated on carious dentine and it was used to evaluate the interface between the modified OB primer and caries-affected dentine. The inhibitory effect of the modified adhesive on caries-affected dentine was studied using in-situ zymography. Results: The fluorometric assay and zymography showed that modified adhesives had high affinity toward both synthetic FRET-peptides and dentine powder substrates, respectively. The immediate micro-tensile bond strength was enhanced for OB (48.0 MPa ± 20.3 SD for BB94 and 42.0 MPa ± 18.7 SD for GM6001) and GB (34.8 MPa ± 19.2 SD for BB94 and 41.7 MPa ± 17.6 SD for GM6001). However, no changes were detected between the control and the inhibitor groups following 3-month storage. Additionally, the micro-permeability of PB and GB showed less dye seepage, to the “hybrid layer” and to the “adhesive”, respectively. The caries-infected dentine was defined significantly by the KHN (< 20.6), AF (> 14.4 A.U.) and by the relative contribution of the mineral (< 36.4%), Porphyrin fluorescence (> 25.3%) and Infected dentine signal (> 0.3%) Raman clusters. The caries-affected dentine-adhesive interface exhibited more hydrophobic resin (32.8% ± 3.9 SD) that maintained over four-week aging. Conclusions: The addition of MMP inhibitors to contemporary dental adhesive systems resulted in modified adhesives that had an enhanced dentine-adhesive interface with inhibited MMP activity. Such properties enhance the clinical performance of adhesive systems.

The use of titanium dental implants in oral rehabilitation has been shown to have a high success rate, Despite this fact, failures do occur, One of the causes of implant failure is peri-implantitis. The aim of this research was to investigate the effect of different surface modifications on titanium surface characteristics, and to explore if one or more of these surface characteristics or antimicrobial agents can reduce microbial adhesion and biofilm formation. Also, to identify major bacterial species associated with peri-implant diseases in human subjects. A variety of chemical and physical surface treatments have been undertaken to explore their effects on titanium surface topography and physicochemical properties. The results showed that physicochemical properties of titanium can be altered with wide range of properties, depending on the type of modification. Investigation of the influence of physicochemical properties of various modified titanium surfaces on the adhesion abilities of different oral bacteria showed that the most important surface characteristic associated with reduced bacterial adhesion to smooth surfaces was hydrophobicity with low surface free energy. However, there was no similar association found for formation of mixed species biofilms on the same surfaces. Functionalizing of titanium surfaces with some antimicrobial natural oils resulted in antiplaque activities similar to those exhibited by chlorhexidine. This suggested that some essential oils could be used in the use of novel antimicrobial agents effective with implant materials. The association of specific bacteria with peri-implant diseases was investigated. These ·results showed that implants were influenced by a wide range of species. Fusobacterium spp., and Prevotella spp. may playa role in initiating peri-implant disease. Other species such as Porphyromonas spp. may then contribute to progression of disease. In conclusion, these studies suggest that bacterial adherence can be reduced by modifying titanium surface properties or by application of anti-bacterial agent. The research also emphasizes the importance of good oral hygiene to prevent accumulation of pathogenic bacteria and thus decrease the risk of peri-implant infections and implant loss.

In vitro models of dental plaque are a valuable tool for understanding the development of plaque-related diseases and assessing potential treatments for these diseases. The main focus of this study was the development of an in vitro model to characterise the changes in bacterial populations from dental plaque associated with health to one associated with gingivitis. By emulating environmental conditions in the oral cavity associated with gingivitis it was possible to see changes in the oral microbiota associated with gingivitis. Using traditional culture techniques the ascendancy of Actinomyces spp. at the expense of Streptococcus spp. was observed with the onset of gingivitis conditions, along with increased proportions of Gram-negative species. To assess the range of cultivable species present isolates, which had previously been cultured in the model, were identified by sequencing of the 16S rRNA gene. After the onset of gingivitis conditions a greater richness of species was identified. Examination of these communities with confocal microscopy and viability staining also revealed structural changes associated with environmental conditions emulating gingivitis. To assess the presence of species which were not frequently identified by culture, but previously shown to be associated with gingivitis, quantitative PCR (qPCR) was used to enumerate Prevotella spp., Fusobacterium spp. and Porphyromonas gingivalis. Prevotella and Fusobacterium spp. were found to be significant members of the microbial communities developed in the CDFF, with Prevotella spp. increasing significantly under conditions emulating gingivitis. Furthermore, the total bacterial counts enumerated by culture were underestimated by approximately 80% compared to the total counts obtained by qPCR. This model was ultimately used to assess the effectiveness of tetracycline, chlorhexidine and silver ion-releasing dental materials against the accumulation of plaque. All of these agents influenced the microbial composition, rather than total microbial numbers, with reduced levels of Actinomyces, Prevotella and Fusobacterium spp. This study has shown that in vitro models of microbial communities associated with health and disease are valuable tools for observing key factors in disease progression. When disease results from changes in the resident microflora the use of such models allows the influence of individual environmental factors to be assessed and also allows the effect of potential treatments on these communities to be examined.

Introduction. The ectopic canine (EC) is a common clinical complication of dental development appearing in 1-2% of the Western population The aetiology is controversial with opinion divided as to a genetic or environmental mechanism. This study addresses the hypothesis that genetic factors play an important role in the aetiology of ectopic maxillary canines. Elucidation of the extent of genetic factors will determine the feasibility of further molecular studies to identify putative genes responsible for ectopic eruption and aid in their identification. Molecular control of tooth eruption would reduce or eliminate the need for surgical procedures associated with buried and impacted teeth and facilitate treatment of those dentofacial anomalies where failure of tooth eruption is a feature. Methods. The study is divided into five parts: 1. A segregation analysis was carried out on 63 pedigrees where a proband was identified as affected with EC, in order to determine whether a genetic component does exist and to provide parameters for further investigation by linkage analysis. 2. Following a positive result from the segregation analysis, linkage analysis was carried out on DNA obtained from an informative, three generation family with seven affected members. 3. Whole exome sequencing was carried out on two distantly related affected members of this family, common, novel and rare variants being identified. 4. The exons and intron-exon junctions of the candidate genes were sequenced using Sanger sequencing in the family and in 18 unrelated cases of EC. 5. In situ hybridisation was carried out using the genes ANO5 and PPP1R14C. Results. Results. The segregation analysis identified a major genetic component with autosomal dominant transmission and the likelihood of a single major locus being involved. The linkage analysis identified several regions of interest and this data was used to filter the results of the exome sequencing. The presence of variations in both PPP1R14C and ANO5 were necessary to precipitate the phenotype. Sanger sequencing of unaffected family members and of unrelated cases showed no similar variants. In situ hybridisation showed both PPP1R14C and ANO5 to be expressed in tooth and supporting tissues, leading to a supposition of digenic inheritance. Conclusion. The genes PPP1R14C and ANO5 are implicated in the aetiology of EC in a digenic inheritance pattern in this family. Further sequencing of affected families and functional studies are required as well as investigation of the methylation status of discordant monozygotic twins.

Astringency is the ‘dry puckering’ sensation attributed to consumption of foods and beverages such as green tea. Previous studies have connected this tactile oral sensation to the aggregation and precipitation of salivary proline-rich proteins (PRPs), yet this mechanism doesn’t occur with all astringent molecules. It also doesn’t consider the impact of astringents directly on lubricating properties of the salivary film and salivary proteins bound to the epithelial layer (mucosal pellicle). Studies of oral epithelial cells have been used to confirm which proteins form the mucosal pellicle. Model particles with different surface chemistries and tissue cultured cell lines have been used to determine how this pellicle forms. These models were then used to decipher effects of green tea polyphenols on the development and composition of the mucosal pellicle. Interfacial rheological measurements were also completed on the pendant drop tensiometer to study the impact of these polyphenols on the development of the saliva film at the air/liquid interface where an elastic protein-rich matrix is formed. The mucosal pellicle is particularly mucin-rich with minimal levels, if any, of PRPs. Epigallocatechin gallate (EGCG), a polyphenol containing a galloyl ring, interacts with MUC5B to a much lower extent than with PRPs. However, this interaction is enough to alter and enhance the binding of MUC5B and other salivary proteins shown to aggregate with polyphenols in pellicle models. This response was echoed with EGCG washes on epithelial cells, indicating improved MUC5B retention as a result of EGCG interactions. Epicatechin (EC) had minimal effects on the salivary pellicle, and shows relatively low levels of interactions with salivary proteins yet is still perceived as astringent. EGCG was also able to alter the interfacial properties of the salivary film to varying degrees among volunteers dependant on salivary protein profiles and concentration. EC however, which has minimal effects except in volunteers with low salivary protein concentration, showed some positive benefits indicating improved wetting properties perhaps due to some surface activity when bound with proteins. Astringency is a multifactorial mechanism, in which protein interactions with polyphenols results in PRP and other salivary protein lead to aggregation, contributing to changes in mucosal pellicle composition and alteration of the interfacial properties of the salivary film. These mechanisms contribute to a dry tactile taste sensation in the mouth.

Introduction: The aim of the study was to investigate novel biomarkers of periodontal disease present in GCF and saliva. Methods: The identities of specific putative biomarkers identified by SELDI-TOF MS were confirmed using 1-D PAGE coupled with LC-MS/MS. These biomarkers were then tested using ELISA in the samples that had been initially screened using SELDI-TOF MS as well as in saliva samples in an independent cohort selected according to diabetic status rather than periodontal status. The effect of non-surgical periodontal therapy, diabetes, saliva sample collection method, eating, time of day, day of the week and storage conditions on the salivary concentration of human neutrophil peptide 1-3 (HNP 1-3), cathelicidin LL-37 and protein S100A8 were tested. Copy number variation for HNP 1-3 gene, DEFA1A3 was tested in an independent cohort and the data analysed for association with presence and degree of periodontal disease. Results: Antimicrobial peptides, HNP 1-3, LL-37 and S100A8 were identified. GCF and salivary HNP 1-3 and LL-37 concentrations were significantly elevated in periodontitis than in gingivitis or health. LL-37 was significantly elevated in aggressive periodontitis (AgP) compared with chronic periodontitis (CP) and gingivitis/health. Periodontal therapy reduced the salivary levels of HNP 1-3. Diabetes, eating and day of week had no significant effect on AMP levels. Saliva collection using spitting method yielded significantly more AMPs than using Salivettes®. Sample collection at 16:00 hours yielded significantly higher amounts of AMPs than samples collected at other times. Sample storage at room temperature (RT) for up to 48 hours had no significant impact on the salivary HNP 1-3 and LL-37 concentration while a proteinase inhibitor did not did improve the recovery of the AMPs after 72 hours.<= 6 copies of DEFA1A3 copy numbers were partly associated with periodontal disease.

Since their introduction to dentistry, calcium silicate based cements have been mainly used for endodontic applications, principally due to their long setting time. Recently, a new formulation of this cement was produced as a coronal restorative material. The aim of this project was to study the nature and dynamics of the interface between this calcium silicate based dental restorative material (BiodentineTM) with human dentine in comparison with glass ionomer cement, and to investigate its capability to induce dentine remineralisation. Different optical, microscopic, and fluorescent labelling techniques have been applied; such as tandem scanning and laser scanning confocal microscopy, which were both used with cement labelling and micropermeability tests to evaluate the interfacial morphology and microscopic appearance along with scanning electron microscopy. Additionally, two-photon fluorescence microscopy was applied in conjugation with Tetracycline labelling to study dentine remineralisation induced by the Biodentine cement; this novel combination provided a useful technique for the observation of mineral formation inside the organic matrix of demineralised dentine when aged in an in-vitro model. Fluorescence lifetime imaging and second harmonic generation imaging were also used for the characterisation of re-mineralisaion and collagen denaturation respectively. For the chemical analysis, Raman spectroscopy was applied to analyse the chemical composition of the cement and the changes associated with its hydration and ageing in different conditions. Micro-Raman imaging was also applied to quantify and model the infiltration of the cement’s hydration products into the dentine. Results indicated an interactive interface between the Biodentine and sound dentine, mediated by the alkaline caustic effect of the cement on the dentine’s organic component, which was associated with mineral transfer, and led to the formation of what we described as a “Mineral Infiltration zone” (MIZ). This later explained the ability of Biodentine to induce remineralisation of dentine and the formation of apatite structures, which indicated the bioactivity of the cement.

In the field of Paediatric Dentistry, brushite cement has potential as an endodontic medicament and bone substitute material. Clinical applications however are limited due to their inherent properties, such as rapid setting time and poor mechanical properties. Antimicrobial e.g. chlorhexidine (CHX) could be incorporated into the cement for localised drug release. Aim and objectives: The aim of this study was to assess if partial replacement of citric acid (CA) by polyacrylic acid (PAA) can improve the properties of conventional brushite cements. The objectives were to assess the effects of varying PAA and CHX concentrations in brushite cements on their setting kinetics, final composition, microstructure of the cement, mechanical properties, degradation and CHX release profile. Materials and Methods: The cements consisted of equimolar β- tricalcium phosphate and monocalcium phosphate monohydrate (β-TCP/MCPM) and 6 or 11% (w/w) CHX. The liquid phase consisted of aqueous 800 mM CA and PAA solution at different ratios. Compositions with no CHX and/or PAA were used as control cements. Setting kinetics and final composition were determined using FTIR and Raman spectroscopy respectively. Brushite microstructure was examined using scanning electron microscopy. The cements were tested for microhardness and biaxial flexural strength (BFS). CHX release was quantified with UV spectroscopy and degradation by mass loss. Results: The setting times for compositions with PAA were delayed by up to 12 hours. FTIR indicated formation of dicalcium citrate and polyacrylate complexes could delay brushite formation. High CHX content inhibited the acid retarding effects and complex formation. Raman mapping demonstrated discrete regions of brushite and CHX in all set cements. Microscopically, PAA addition resulted in denser and less porous structure. The BFS ranged from 5.8 ± 1.3 MPa to 11.1 ± 1.2 MPa. CHX incorporation resulted in reduced BFS and modulus whilst PAA addition increased it. The average mass change was significantly different between compositions with and with no chlorhexidine; 12% and 0.2% respectively at the end of study period. The daily degradation rate ranged from 0.1 ± 0.03 wt% to 0.6 ± 0.15 wt%. PAA presence reduced CHX release from more than 90% to less than 20% over 4 weeks. Conclusion: PAA substantially slowed the setting reaction and chlorhexidine release characteristics, altered the final brushite crystal microstructure, increased mechanical properties but did not affect the degradation kinetics.

Tooth enamel is built of crystalline calcium phosphate fixed into a protein matrix. When acid comes into contact with tooth surface it encourages demineralisation and erosion of tooth enamel. Eroded tooth with small cavities becomes vulnerable to the attack of microflora present in dental plaque on tooth surfaces and increases the risk of tooth decay. It has been reported that dental erosion and caries among children and adults have an increasing trend. It is thought that changes in drinking habit and more interest in consumption of soft drinks could be a possible reason for this dental health problem. To reduce the risk of dental erosion several studies were carried out and some research indicated that milk proteins and peptides have anti-erosive and anti-caries effect. In this category casein phosphopeptides (CPP) a tryptic hydrolysate of casein was widely studied to reduce dental problems. In addition, glycomacropeptide (GMP), a hydrophilic part of κ-casein released in whey, was of interest. In this study, attempts were made to reduce the mineral dissolution of enamel using GMP and GMP fractions during different acid contact times. To simulate the mineral dissolution of enamel hydorxyapatite (HA, calcium phosphate) was used. At different experiments HA was treated with GMP prior to exposure to 0.1M citrate buffer at different pH levels of 2.5-4.5. The level of dissolved calcium and phosphate into citrate buffer was measured by atomic absorption spectroscopy and Allen‘s colorimetric method, respectively. The erosive effect of soft drinks was investigated using orange juice and coca-cola containing GMP and its protection level was determined by measuring the amount of dissolved minerals in soft drinks compared to a control. To examine the protective effect of the GMP from acid attack on enamel surface, human enamel was studied by scanning electron microscopy. This study showed that GMP could reduce dental erosion and act better than CPP at low pH (3.0). This protective effect of GMP against mineral dissolution of enamel could be due to its attachment to the surface, its amino acid sequence and glycosylation level and/or to the overall net charge of the peptide.

The Tn916-like family of conjugative transposons are broad host range mobile genetic elements and are clinically important as they are one of the major vectors responsible for the spread of antibiotic resistance among bacterial pathogens. This study was designed to investigate the behaviour of the conjugative transposons Tn916 and Tn5397, focusing on the transcriptional regulation and transfer. The proposed regulatory system of Tn916 involves transcriptional attenuation upstream of tet(M) and is regulated by tetracycline. The translation of orf12 is central to this regulatory mechanism as it is the translating ribosome upon orf12 RNA that is hypothesised to destroy, or prevent the formation of the transcriptional terminators. This hypothesis was tested using a Bacillus subtilis construct, with a 2 bp mutation disrupting the start codon of orf12. This construct is expected to result in the transcriptional terminators being permanently formed as the ribosome will no longer translate the orf12 and destroy them. Results indicate a lower transcription of tet(M) and downstream genes which was supported by the slower growth rate of the B. subtilis mutant compared to the wild type upon challenged with tetracycline. When tetracycline is present, a reduced fitness of this mutant was observed compared to the wild type. However, the transfer frequency of the B. subtilis mutant was similar to that of the wild type. The transcription of Tn916 and Tn5397 was investigated by quantifying the expression level using reporter assays, where the Ptet(M) promoter and the open reading frames upstream of tet(M) were cloned upstream of a ß-glucuronidase gene. In the presence of tetracycline, Tn916 wild type construct was upregulated whereas the Tn5397 wild type construct showed a constant expression level. Disruption of the start codon of orf12 (Tn916) and orf26 (Tn5397) has also led to a constant expression level of ß-glucuronidase. The termination efficiency of the Tn916 terminators was estimated using promoter assays and a published algorithm. Results suggest that the large terminator is more efficient [47% (± 18)] than the small terminator [23% (± 15)], which was supported by the algorithm analysis for Tn916. Finally, reciprocal gene transfer of Tn5397 between Clostridium difficile and Enterococcus faecalis was demonstrated. The transfer frequency [± standard deviation (SD)] detected was 8.85 x 10-8 (± 2.14 x 10-7) per recipient. Tn5397 integrates into the genome of E. faecalis at a single site that is within an orf encoding the phosphotransferase (PTS) IIA component. Comparative growth curves showed that the acquisition of Tn5397 has a very small effect on the growth of E. faecalis. This work has extended the current knowledge of the regulation and transfer of conjugative transposons of the Tn916-like family. It has provided a better understanding about the mechanism of transcriptional regulation of these elements.