Jennifer Kirkham

The Chemistry of Enamel Caries

The chemical changes which occur during the process of carious destruction of enamel are complex due to a number of factors. First, substituted hydroxyapatite, the main component of dental enamel, can behave in a very complex manner during dissolution. This is due not only to its ability to accept substituent ions but also to the wide range of calcium phosphate species which can form following dissolution. In addition, the composition, i.e., the extent of substitution, changes throughout enamel in the direction of carious attack, i.e., from surface to interior. Both surface and positively birefringent zones of the lesion clearly illustrate that carious destruction is not simple dissolution. Selective dissolution of soluble minerals occurs, and there is the probability of reprecipitation. The role of fluoride here is crucial in that not only does it protect enamel per se but also its presence in solution means that rather insoluble fluoridated species can form very easily, encouraging redeposition. The role of organic material clearly needs further investigation, but there is the real possibility of both inhibition of repair and facilitation of redeposition. For the future, delivering fluoride deep into the lesion would appear to offer the prospect of improved repair. This would entail a delivery vehicle which solved the problem of fluoride uptake by apatite at the tooth surface. Elucidation of the role of organic material may also reveal putative mechanisms for encouraging repair and/or protecting the enamel mineral.

ARCHITECTURE OF INTACT NATURAL HUMAN PLAQUE BIOFILMS STUDIED BY CONFOCAL LASER SCANNING MICROSCOPY

Determination of the structure of human plaque will be of great benefit in the prediction of its formation and also the effects of treatment. However, a problem lies in the harvesting of undisturbed intact plaque samples from human volunteers and the viewing of the biofilms in their natural state. In this study, we used an in situ device for the in vivo generation of intact dental plaque biofilms on natural tooth surfaces in human subjects. Two devices were placed in the mouths of each of eight healthy volunteers and left to generate biofilm for 4 days. Immediately upon removal from the mouth, the intact, undisturbed biofilms were imaged by the non-invasive technique of confocal microscopy in both reflected light and fluorescence mode. Depth measurements indicated that the plaque formed in the devices was thicker round the edges at the enamel/nylon junction (range = 75-220 μm) than in the center of the devices (range = 35-215 μm). The reflected-light confocal images showed a heterogeneous structure in all of the plaque biofilms examined; channels and voids were clearly visible. This is in contrast to images generated previously by electron microscopy, suggesting a more compact structure. Staining of the biofilms with fluorescein in conjunction with fluorescence imaging suggested that the voids were fluid-filled. This more open architecture is consistent with recent models of biofilm structure from other habitats and has important implications for the delivery of therapeutics to desired targets within the plaque.

Self-assembling Peptide Scaffolds Promote Enamel Remineralization

Rationally designed β-sheet-forming peptides that spontaneously form three-dimensional fibrillar scaffolds in response to specific environmental triggers may potentially be used in skeletal tissue engineering, including the treatment/prevention of dental caries, via bioactive surface groups. We hypothesized that infiltration of caries lesions with monomeric low-viscosity peptide solutions would be followed by in situ polymerization triggered by conditions of pH and ionic strength, providing a biomimetic scaffold capable of hydroxyapatite nucleation, promoting repair. Our aim was to determine the effect of an anionic peptide applied to caries-like lesions in human dental enamel under simulated intra-oral conditions of pH cycling. Peptide treatment significantly increased net mineral gain by the lesions, due to both increased remineralization and inhibition of demineralization over a five-day period. The assembled peptide was also capable of inducing hydroxyapatite nucleation de novo. The results suggest that self-assembling peptides may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

AN IN VITRO STUDY OF THE USE OF PHOTODYNAMIC THERAPY FOR THE TREATMENT OF NATURAL ORAL PLAQUE BIOFILMS FORMED IN VIVO

Seven-day oral plaque biofilms have been formed on natural enamel surfaces in vivo using a previously reported in situ device. The devices are then incubated with a cationic Zn(II) phthalocyanine photosensitizer and irradiated with white light. Confocal scanning laser microscopy (CSLM) of the biofilms shows that the photosensitizer is taken up into the biomass of the biofilm and that significant cell death is caused by photodynamic therapy (PDT). In addition, the treated biofilms are much thinner than the control samples and demonstrate a different structure from the control samples, with little evidence of channels and a less dense biomass. Transmission electron microscopy (TEM) of the in vivo-formed plaque biofilms reveals considerable damage to bacteria in the biofilm, vacuolation of the cytoplasm and membrane damage being clearly visible after PDT. These results clearly demonstrate the potential value of PDT in the management of oral biofilms.

The Effect of Fluoride on the Developing Tooth

This review aims to outline the effects of fluoride on the biological processes involved in the formation of tooth tissues, particularly dental enamel. Attention has been focused on mechanisms which, if compromised, could give rise to dental fluorosis. The literature is extensive and often confusing but a much clearer picture is emerging based on recent more detailed knowledge of odontogenesis. Opacity, characteristic of fluorotic enamel, results from incomplete apatite crystal growth. How this occurs is suggested by other changes brought about by fluoride. Matrix proteins, associated with the mineral phase, normally degraded and removed to permit final crystal growth, are to some extent retained in fluorotic tissue. Fluoride and magnesium concentrations increase while carbonate is reduced. Crystal surface morphology at the nano-scale is altered and functional ameloblast morphology at the maturation stage also changes. Fluoride incorporation into enamel apatite produces more stable crystals. Local supersaturation levels with regard to the fluoridated mineral will also be elevated facilitating crystal growth. Such changes in crystal chemistry and morphology, involving stronger ionic and hydrogen bonds, also lead to greater binding of modulating matrix proteins and proteolytic enzymes. This results in reduced degradation and enhanced retention of protein components in mature tissue. This is most likely responsible for porous fluorotic tissue, since matrix protein removal is necessary for unimpaired crystal growth. To resolve the outstanding problems of the role of cell changes and the precise reasons for protein retention more detailed studies will be required of alterations to cell function, effect on specific protein species and the nano-chemistry of the apatite crystal surfaces.

Penetration of Fluoride into Natural Plaque Biofilms

Caries occurs at inaccessible stagnation sites where plaque removal is difficult. Here, the penetration through plaque of protective components, such as fluoride, is likely to be crucial in caries inhibition. We hypothesized that topically applied fluoride would readily penetrate such plaque deposits. In this study, plaque biofilms generated in vivo on natural enamel surfaces were exposed to NaF (1000 ppm F−) for 30 or 120 sec (equivalent to toothbrushing) or for 30 min. Biofilms were then sectioned throughout their depth, and the fluoride content of each section was determined with the use of a fluoride electrode. Exposure to NaF for 30 or 120 sec increased plaque fluoride concentrations near the saliva interface, while concentrations near the enamel surface remained low. Fluoride penetration increased with duration of NaF exposure. Removal of exogenous fluoride resulted in fluoride loss and redistribution. Penetration of fluoride into plaque biofilms during brief topical exposure is restricted, which may limit anti-caries efficacy.

Mutations in CNNM4 Cause Jalili Syndrome, Consisting of Autosomal-Recessive Cone-Rod Dystrophy and Amelogenesis Imperfecta

The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.

Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta

Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca2+-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation.

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned λpr promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after ‘collision’ has caused their arrest.

In vitro Studies of the Penetration of Adhesive Resins into Artificial Caries–Like Lesions

Instead of removing the porous carious tissue at a relatively late stage in the disease process, attempts have been made to ‘fill’ the microporosities of lesions at a much earlier stage of lesion development. This would not only reduce the porosity and therefore access of acid and egress of dissolved material, but also afford some mechanical support to the tissue and perhaps inhibit further attack. Successful infiltration of materials into lesions has been demonstrated previously using resorcinol–formaldehyde which, however, was clinically unacceptable. The advent of dental adhesives with potentially suitable properties has prompted a re–examination of this concept. Artificial lesions of enamel were generated in extracted human teeth using acidified gels. A range of currently available adhesive materials was then used to infiltrate the porosities. The extent of occlusion of the lesion porosities was determined both qualitatively using light microscopy and quantitatively using a chloronaphthalene imbibition technique. The effect of such treatment upon subsequent exposure to acid gels was also investigated. Results showed that up to 60% of the lesion pore volume had been occluded following infiltration with some of the materials and that this treatment was capable of reducing further acid demineralization. The development of such treatment strategies could offer potential noninvasive means of treating early enamel lesions.