Simon R. Wood

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.

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 SUBCELLULAR LOCALIZATION OF ZN(II) PHTHALOCYANINES AND THEIR REDISTRIBUTION ON EXPOSURE TO LIGHT

ABSTRACT

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.

A Comparative Study of the Cellular Uptake and Photodynamic Efficacy of Three Novel Zinc Phthalocyanines of Differing Charge

Abstract— Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20‐tetra(m‐hydroxyphenyl)chlorin (m‐THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF‐1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly unproved activity over PHP and m‐THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m‐THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low‐level light exposure, and this was charge dependent The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zhic [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low‐level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.

The characterisation of three substituted zinc phthalocyanines of differing charge for use in photodynamic therapy. A comparative study of their aggregation and photosensitising ability in relation to mTHPC and polyhaematoporphyrin

Three substituted zinc (II) phthalocyanines (one anionic, one cationic and one hydrophobic) have been compared to two clinically used photosensitisers, 5,10,15,20-tetra (m-hydroxyphenyl) chlorin (mTHPC) and polyhaematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Oxygen-consumption experiments, performed to follow the photo-oxidation of tryptophan, histidine and bovine serum albumin (BSA), suggest that the anionic phthalocyanine is the most efficient photosensitiser. The efficacy of BSA oxidation is much greater than that of tryptophan or histidine, which is partly due to monomerisation of the sensitisers upon binding to BSA. Spectra recorded in aqueous solution reveal that all five compounds are highly aggregated, but monomerisation is induced upon the addition of BSA or methanol. Using a range of methanol-buffer solutions, the aggregation state has been directly related to the efficacy of tryptophan photo-oxidation with maximal rates of oxidation achieved when the sensitiser is monomeric. Using erythrocytes as a simple membrane model, the efficacy of each sensitiser exhibits a different trend from that predicted by oxygen-consumption experiments. The anionic phthalocyanine is the least effective at photohaemolysis, whereas the cationic and hydrophobic phthalocyanines have improved activity over PHP and mTHPC.

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.

Evidence for Charge Domains on Developing Enamel Crystal Surfaces

The control of hydroxyapatite crystal initiation and growth during enamel development is thought to be mediated via the proteins of the extracellular matrix. However, the precise nature of these matrix-mineral interactions remains obscure. The aim of the present study was to use a combination of atomic and chemical force microscopy to characterize developing enamel crystal surfaces and to determine their relationship with endogenous enamel matrix protein (amelogenin). The results show regular and discrete domains of various charges or charge densities on the surfaces of hydroxyapatite crystals derived from the maturation stage of enamel development. Binding of amelogenin to individual crystals at physiological pH was seen to be coincident with positively charged surface domains. These domains may therefore provide an instructional template for matrix-mineral interactions. Alternatively, the alternating array of charge on the crystal surfaces may reflect the original relationship with, and influence of, matrix interaction with the crystal surfaces during crystal growth.