Steven J. Brookes

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.

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.

Enamel matrix proteins; old molecules for new applications

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.

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.

Characterization of a Porcine Amelogenin Preparation, EMDOGAIN, a Biological Treatment for Periodontal Disease

EMDOGAIN is derived from porcine developing enamel matrix and has been shown to facilitate regeneration of the periodontium, although its mechanism of action is unknown. The aim of the present study was to identify enamel matrix proteins and proteolytic enzymes present in EMDOGAIN and compare them with those extracted from developing porcine enamel itself. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and zymography were used to identify the proteins present and to determine their enzyme activity. The results showed that developing enamel contained amelogenins, albumin, amelin, and enamelin. EMDOGAIN, however, contained only amelogenins. Both metalloendoproteases and serine protease activity were revealed in both EMDOGAIN and developing enamel. The roles of the amelogenin and enzyme components, if any, in periodontal regeneration are unknown.

Mutations in C4orf26, encoding a peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the proteins phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.

Physico-chemical properties of crystal surfaces in matrix-mineral interactions during mammalian biomineralisation

Surfaces of developing enamel crystals were shown to comprise of alternating domains of positive and less positive (perhaps even negative) charge density which directly bind a number of matrix proteins via electrostatic interactions. Studies using synthetic mineral crystals demonstrated stereo-specific docking of charged residues with crystal lattice sites. Preferential binding of matrix proteins to specific crystal faces related to interfacial hydrophobicity/hydrophobicity, was shown to control crystal habit.

A Method for the Quantitative Site-Specific Study of the Biochemistry within Dental Plaque Biofilms Formed in vivo

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.