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Heimler syndrome (HS) consists of recessively inherited sensorineural hearing loss, amelogenesis imperfecta (AI) and nail abnormalities, with or without visual defects. Recently HS was shown to result from hypomorphic mutations in PEX1 or PEX6, both previously implicated in Zellweger Syndrome Spectrum Disorders (ZSSD). ZSSD are a group of conditions consisting of craniofacial and neurological abnormalities, sensory defects and multi-organ dysfunction. The finding of HS-causing mutations in PEX1 and PEX6 shows that HS represents the mild end of the ZSSD spectrum, though these conditions were previously thought to be distinct nosological entities. Here, we present six further HS families, five with PEX6 variants and one with PEX1 variants, and show the patterns of Pex1, Pex14 and Pex6 immunoreactivity in the mouse retina. While Ratbi et al. found more HS-causing mutations in PEX1 than in PEX6, as is the case for ZSSD, in this cohort PEX6 variants predominate, suggesting both genes play a significant role in HS. The PEX6 variant c.1802G>A, p.(R601Q), reported previously in compound heterozygous state in one HS and three ZSSD cases, was found in compound heterozygous state in three HS families. Haplotype analysis suggests a common founder variant. All families segregated at least one missense variant, consistent with the hypothesis that HS results from genotypes including milder hypomorphic alleles. The clinical overlap of HS with the more common Usher syndrome and lack of peroxisomal abnormalities on plasma screening suggest that HS may be under-diagnosed. Recognition of AI is key to the accurate diagnosis of HS., Competing Interests: The authors declare no conflict of interest.

We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation (c.586C>A, p.P196T) in the I-domain of integrin-β6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell-matrix interaction or compromised ITGB6 activation of transforming growth factor-β (TGF-β) impacting indirectly on ameloblast-ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell-matrix interactions during enamel formation.

The conventional approach to identifying the defective gene in a family with an inherited disease is to find the disease locus through family studies. However, the rapid development and decreasing cost of next generation sequencing facilitates a more direct approach. Here, we report the identification of a frameshift mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta (AI). Whole-exome sequencing of three affected family members and subsequent filtering of shared variants, without prior genetic linkage, sufficed to identify the pathogenic variant. Simultaneous analysis of multiple family members confirms segregation, enhancing the power to filter the genetic variation found and leading to rapid identification of the pathogenic variant. LAMB3 encodes a subunit of Laminin-5, one of a family of basement membrane proteins with essential functions in cell growth, movement and adhesion. Homozygous LAMB3 mutations cause junctional epidermolysis bullosa (JEB) and enamel defects are seen in JEB cases. However, to our knowledge, this is the first report of dominant AI due to a LAMB3 mutation in the absence of JEB.

Objective: Enamel matrix derivative (EMD), is an extract of porcine developing enamel matrix. Its commercialised form Emdogain, is claimed to stimulate periodontal regeneration by recapitulating original developmental processes, although the mechanism remains unclear. Our objective was to investigate interactions between EMD and human periodontal ligament (HPDL) fibroblasts in vitro., Design: HPDL fibroblasts were cultured in the presence of fluorescently labelled EMD and cellular EMD uptake was monitored using confocal laser scanning microscopy and immunohistochemistry. Internalised EMD proteins were characterised using SDS-PAGE., Results: EMD was internalised by HPDL fibroblasts leading to the appearance of multiple, vesicle-like structure in the cytoplasm. The internalised protein was composed mainly of the major 20kDa amelogenin component of EMD which was subsequently processed with time to generate a cumulative 5kDa component., Conclusions: Cellular uptake and subsequent intracellular processing of EMD components by dental mesenchymal cells may play a role in EMD bioactivity and in part explain the turnover of Emdogain when placed clinically., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The prokaryote Corynebacterium matruchotii produces calcium phosphate (bone salt) and may serve as a convenient model for examining individual factors relevant to vertebrate calcification. A factor of current clinical uncertainty is silicon. To investigate its possible role in biomineralisation advanced optical (digital deconvolution and 3D fluorescent image rendering) and electron microscopy (EDX microanalysis and elemental mapping) were applied to calcifying microbial colonies grown in graded Si concentrations (0-60mM). Cell viability was confirmed throughout by TO-PRO-3-iodide and SYTO-9 nucleic acid staining. It was observed that calcium accumulated in dense intracellular microspherical objects (types i-iii) as nanoparticles (5 nm, type i), nanospheres (30-50 nm, type ii) and filamentous clusters (0.1-0.5 μm, type iii), with a regular transitory Si content evident. With bacterial colony development (7-28 days) the P content increased from 5 to 60%, while Si was displaced from 60 to 5%, distinguishing the phenomenon from random contamination, and with a significant relationship (p<0.001) found between calcified object number and Si supplementation (optimum 0.01mM). The Si-containing, intracellular calcified objects (also positive for Mg and negative with Lysensor blue DND-167 for acidocalcisomes) were extruded naturally in bubble-like chains to complete the cycle by coating the cell surface with discrete mineral particles. These could be harvested by lysis, French press and density fractionation when Si was confirmed in a proportion. It was concluded that the unexplained orthopaedic activity of Si may derive from its special property to facilitate calcium phosphorylation in biological systems, thereby recapitulating an ancient and conserved bacterial cycle of calcification via silicification., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Mutations in WDR72 have been identified in autosomal recessive hypomaturation amelogenesis imperfecta (AI)., Objective: to describe a novel WDR72 mutation and report the ultrastructural enamel phenotype associated with a different WDR72 mutation., Methods: A family segregating autosomal recessive hypomaturation AI was recruited, genomic DNA obtained and WDR72 sequenced. Four deciduous teeth from one individual with a previously published WDR72 mutation, extracted as part of clinical care, were subjected to scanning electron microscopy, energy-dispersive X-ray analysis and transverse microradiography., Results: A novel homozygous nonsense mutation, R897X, was identified in WDR72 in a family originating from Pakistan. Ultrastructural analysis of enamel from the deciduous teeth of an AI patient with the WDR72 mutation S783X revealed energy-dispersive X-ray analysis spectra with normal carbon and nitrogen peaks, excluding retention of enamel matrix protein. However, transverse microradiography values were significantly lower for affected teeth when compared to normal teeth, consistent with reduced mineralisation. On scanning electron microscopy the enamel rod form observed was normal, yet with inter-rod enamel more prominent than in controls. This appearance was unaltered following incubation with either α-chymotrypsin or lipase., Conclusions: The novel WDR72 mutation described brings the total reported WDR72 mutations to four. Analyses of deciduous tooth enamel in an individual with a homozygous WDR72 mutation identified changes consistent with a late failure of enamel maturation without retention of matrix proteins. The mechanisms by which intracellular WDR72 influences enamel maturation remain unknown., (Copyright © 2010 S. Karger AG, Basel.)

Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y --> H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein-protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI.

The periosteum and Sharpey's fibre extensions occupy the musculoskeletal interface and may be strategic in age-related deterioration. Because of its exceptionally powerful insertions the porcine mandible is an ideal model and its periosteal system was compared in 4 separate regions of adult young (1 year) and older (3 year) animals. These were examined by undecalcified histology, collagen immunohistochemistry and mineral histochemistry using polarization, epifluorescence and laser confocal microscopy; mineral ultrastructure was facilitated by chromium labelling with EDX microanalysis. Birefringent Sharpey's fibres were coarse (>8 microm) or fine and classified as horizontal (more common with age), oblique (most common in youth) or vertical (least common); in addition they were designated "superficial", "transcortical" and "intertrabecular" (the latter being deep, coarse and vertical). Their specific affinity for collagen type III FITC-labelled antibody demonstrated 3-dimensional arrays of bone-permeating fibres. With age at each region the cortical thickness rose (e.g. 4.9 mm to 9.3 mm), the periosteum thinned (e.g. 180-/+7 microm to 129-/+8 microm; p<0.001), and the periosteum: bone ratio diminished (e.g. 3.65-/+0.36 to 1.40-/+0.14; p<0.001) while Sharpey's fibres became fewer, fragmented, superficial and shortened (e.g. 226-/+27 microm to 55-/+6 microm; p<0.001). Accompanying was the sporadic encroachment of calcified particles, 1 microm diameter, in irregular periosteal aggregates or interlinked around Sharpey bundles (resembling calcifying turkey leg tendon). EDX microanalysis confirmed prominent chromium spectral peaks in the older periosteum only, coincident with chromium-labelled mineral "ghosts". It was concluded that the periosteum and Sharpey's fibres, deep-penetrating and complex in youth, partially hardens and regresses with age with implications for its functional properties.

In a group of families in northern Sweden, a mutation in the ENAM gene (predicted to produce a highly truncated protein) results in the local hypoplastic form of autosomal dominant amelogenesis imperfecta. In this study, sections of deciduous teeth from members of 3 of these families were examined by scanning electron microscopy (SEM) and the enamel mineral was analysed by energy dispersive X-ray spectroscopy (EDX). The sections were also probed with antibodies raised to a conserved sequence of the enamelin protein. Selected intact teeth were first analysed by digital imaging and ascribed with an 'Enamel Defects Index' (EDI) score. SEM of tooth sections revealed disrupted prism morphology and the prisms had a glass-like appearance in some areas. These areas of dysplasia were sometimes irregular but formed regular arrays in others. Comparison of EDI scores with SEM indicated that in one tooth the surface had no measurable defects but significant defects were present in the underlying enamel microstructure. SEM immunohistochemistry with the antibody raised to a fragment of the enamelin protein produced positive, but light, labelling throughout normal enamel. In dysplastic areas, however, the labelling intensity appeared to be reduced. The results indicate that the presence of functional enamelin in the correct amounts is necessary for correct prism morphogenesis. In addition, a combination of EDI and structural analysis indicate that defects in enamel microstructure are not necessarily visible as defects on the surface of the tooth, suggesting the possibility, at least, that some instances of under-diagnosis may occur.

Background: Nonsense mutations in FAM83H are a recently described underlying cause of autosomal dominant (AD) hypocalcified amelogenesis imperfecta (AI)., Objective: This study aims to report a novel c.1374C>A p.Y458X nonsense mutation and describe the associated ultrastructural phenotype of deciduous teeth., Methods: A family of European origin from the Iberian Peninsula with AD-inherited AI was ascertained. Family members were assessed through clinical examination and supporting investigations. Naturally exfoliated deciduous teeth from 2 siblings were investigated by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and transverse microradiography (TMR)., Results: On clinical and radiographic investigation the appearances of the affected deciduous and permanent teeth were consistent with hypocalcified AI with small focal areas of more normal looking enamel. DNA sequencing identified a novel c.1374C>A p.Y458X FAM83H nonsense mutation in affected, but not in either unaffected family members or unrelated controls. Exfoliated teeth were characterised by substantial post-eruptive enamel loss on gross examination. Irregular, poor quality enamel prisms were observed on SEM. These were coated in amorphous material. TMR and EDX confirmed reduced mineral and increased organic content in enamel, respectively., Conclusions: FAM83H nonsense mutations have recently been recognised as a cause of AD hypocalcified AI. We report a novel nonsense FAM83H mutation and describe the associated preliminary ultrastructural phenotype in deciduous teeth. This is characterised by poorly formed enamel rods with inappropriate retention of amorphous material, which is likely to represent retained organic matrix that contributes to the overall hypomineralised phenotype., (2009 S. Karger AG, Basel.)

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 beta 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.

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.

Nectin-1 is a member of a sub-family of immunoglobulin-like adhesion molecules and a component of adherens junctions. In the current study, we have shown that mice lacking nectin-1 exhibit defective enamel formation in their incisor teeth. Although the incisors of nectin-1-null mice were hypomineralized, the protein composition of the enamel matrix was unaltered. While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1-null mice correlated with separation of the cell layers at this interface. Numerous, large desmosomes were present at this interface in wild-type mice; however, where adhesion persisted in the mutant mice, the desmosomes were smaller and less numerous. Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly. Importantly, our results show that integrity of the SI-ameloblast interface is essential for normal enamel mineralization.

We have used synchrotron X-ray diffraction to study the texture and the change in lattice parameter as a function of position in a cross section of human dental enamel. Our study is the first to map changes in preferred orientation and lattice parameter as a function of position within enamel across a whole tooth section with such high resolution. Synchrotron X-ray diffraction with a micro-focused beam spot was used to collect two-dimensional (2D) diffraction images at 150 microm spatial resolution over the entire tooth crown. Contour maps of the texture and lattice parameter distribution of the hydroxyapatite phase were produced from Rietveld refinement of diffraction patterns generated by azimuthally sectioning and integrating the 2D images. The 002 Debye ring showed the largest variation in intensity. This variation is indicative of preferred orientation. Areas of high crystallite alignment on the tooth cusps match the expected biting surfaces. Additionally we found a large variation in lattice parameter when travelling from the enamel surface to the enamel-dentine junction. We believe this to be due to a change in the chemical composition within the tooth. The results provide a new insight on the texture and lattice parameter profiles within enamel.

Rationally designed beta-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.

The architecture of microbial biofilms especially the outer regions have an important influence on the interaction between biofilm and local environment particularly on the flux of materials into and out of biofilm compartments and as a consequence, biofilm metabolic behaviour. In the case of dental plaque biofilms, architecture will determine access of nutrients including acidogenic substrates and therapeutic materials to the microbial biomass and to the underlying tooth surface. Manipulation of this architecture may offer a means of altering mass transfer into the whole biofilm and biomass and raises the possibility of improving access of therapeutics. Plaque biofilms formed in vivo on human enamel were subjected to a number of different chemical conditions while under observation by confocal laser scanning microscopy in reflection mode. In this way the outer 50-100 microm or so of the biofilms was examined. Density and distribution of biomass were recorded as degree of reflectance. The amount and density of biofilm biomass increased from the plaque saliva interface towards the interior. Plaque biofilms were robust and little affected by mechanical manipulation, high ionic strength or low pH (2.5). Detergent (SLS), however, often appeared to either remove biomass and/or dramatically reduce its density.

Increasing evidence suggests that amelogenin, long held to be a structural protein of developing enamel matrix, may also have cell signaling functions. However, a mechanism for amelogenin cell signaling has yet to be described. The aim of the present study was to use dynamic chemical force spectroscopy to measure amelogenin interactions with possible target cells. Full-length amelogenin (rM179) was covalently attached to silicon nitride AFM tips. Synthetic RGD peptides and unmodified AFM tips were used as controls. Amelogenin-RGD cell binding force measurements were carried out using human periodontal ligament fibroblasts (HPDF) from primary explants and a commercially available osteoblast-like human sarcoma cell line as the targets. Results indicated a linear logarithmic dependence between loading rate and unbinding force for amelogenin-RGD target cells across the range of loading rates used. For RGD controls, binding events measured at 5.5 nN s-1 force loading rate resulted in a mean force of 60 pN. Values for amelogenin-fibroblast and amelogenin-osteoblast-like cell unbinding forces, measured at similar loading rates, were 50 and 55 pN, respectively. These data suggest that amelogenin interacts with potential target cells with forces characteristic of specific ligand-receptor binding, suggesting a direct effect for amelogenin at target cell membranes.

Enamel matrix comprises nanospheres predominantly composed of amelogenin. Studies have shown that recombinant amelogenin forms nanospheres similar to those formed in vivo, but it is unclear exactly how nanospheres assemble in vivo. Are amelogenin monomers secreted into the enamel matrix where they then self-assemble to form nanospheres, or does nanosphere assembly actually occur intracellularly? The aim of this study was to attempt to answer this question. Rat enamel organs were treated with the bifunctional cross-linker, dithio bis (succinimidyl propionate) (DSP), which cross-links primary amines lying in close molecular proximity. The key to this technique is the fact that DSP cross-links are later sensitive to reductive cleavage. The cross-linked proteins were first subjected to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and then to reducing SDS-PAGE in the second dimension (so-called diagonal electrophoresis) followed by western blot probing with anti-amelogenin. The results indicated that intracellular amelogenin monomers are in close neighbor contact, forming complexes comprising up to six individual amelogenin monomers. We suggest that these initial complexes are prefabricated intracellularly before secretion. Once secreted, these prefabricated subunits assemble further to form the mature full-size nanospheres containing hundreds of individual amelogenins characteristic of enamel matrix.

The effect of bisphosphonates on trabecular microarchitecture may contribute to the reduced risk of vertebral fracture with treatment independent of the bone volume. Trabecular structure was examined at the twelfth thoracic vertebra after 2 years of treatment of two groups of ovariectomized baboons on high and low doses of alendronate, compared with ovariectomized and non-ovariectomized controls. Standard 2D histological measurements showed that alendronate treatment of ovariectomized animals resulted in significantly higher total trabecular length and a lower marrow star volume in comparison with ovariectomized controls indicating preservation of connectivity. Similarly when the vertebrae were examined using a novel thick slice technique that combines 2D and 3D information, ovariectomy produced a significantly higher number of "real" trabecular termini in comparison with normal. When ovariectomized animals were treated with increasing doses of alendronate, fewer "real" termini were seen. MicroCT analysis (2D and 3D) correlated well with the histological measurements, although more variability and less discrimination between groups was seen, with no statistically significant differences with alendronate treatment. Reduced vertebral fracture risk with alendronate may be due to a combination of factors including the increased bone volume, reduced turnover and greater mineralization reported by others. Added to this is now suggested the preservation of several aspects of vertebral cancellous architecture, with microscopy the most sensitive method of analysis.

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.

Apatite crystal growth rate and morphology in mineralized tissues are considered to be controlled by surface interaction with extracellular matrix proteins. During enamel maturation where protein is finally removed from crystal surfaces to permit massive crystal growth, pH oscillates between approximately 5.8 and approximately 7.2. With this in mind, a study of enamel apatite surface chemistry in terms of local environmental pH was undertaken. Using atomic force microscopy adhesion force measurements were made between hydroxylated or carboxylated cantilever tips and maturation stage crystals between pH 2 and 10. Adhesion force increased from pH 10 to a maximum at pH 6.6 presumably due to increased hydrogen bonding due to replacement of surface cations (Na, Ca, Mg) with protons and/or protonation of phosphate per se. Below pH 6.6 adhesion force decreased and became very variable indicating that the surface had become unstable probably due to removal of fully protonated phosphate from the surface by adherence to the cantilever tip. Frictional force measurements also revealed 2-3, approximately 30 nm diameter high friction domains in bands across the crystal long axis. Their location mirrored the binding pattern of similarly sized amelogenin aggregates seen in vitro. The data suggests that specific protein binding sites may exist on crystal surfaces and may be released at lower pH by protonation which would lower cationic charge on both crystal surface and ionic charge on the protein. Instability of the crystal surface could also play a role.

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., (Copyright 2004 S. Karger AG, Basel)

Aims: To determine whether the stringency of sterilization procedures for biological components of in vivo dental plaque-generating devices based on enamel can be increased to minimize prion risk without compromising natural biofilm composition., Methods and Results: The composition of in vitro biofilms, grown on hypochlorite-treated and untreated autoclaved enamel surfaces, was determined using culture-based methods and checkerboard DNA: DNA hybridization analysis. No differences were found between biofilms recovered from either substrate., Significance: Several in situ models allow generation of plaque in the oral cavity, followed by recovery of intact biofilms for experimentation. Approaches allowing plaque formation on natural tooth surfaces are most valuable, but present a possible infection risk to volunteers wearing plaque-collecting devices, particularly with respect to prions. Hypochlorite treatment of biological material, as an adjunct to autoclaving, reduces infection risk without compromising biofilm composition and should be adopted in all future studies using plaque-generating devices incorporating enamel, where there is a potential prion threat, and further investigated in other biological hard tissues.

Cu2+ is cariostatic in rats reportedly due to it bacteriocidal properties. Here, we report the use of a simple abiotic model system to investigate whether Cu2+ has any inhibitory effect on the acid dissolution of human enamel. Crowns were exposed to a sequence of seven 10 mmol/l acetic acid challenges. The mineral dissolved during each challenge was then determined. CuSO4 (10 mmol/l) was present during the fourth of these challenges. Loss of calcium and phosphate were reduced by 57 and 63%, respectively, (P<0.0001) in the presence of Cu2+. Losses were also significantly reduced during the next acidic challenge in the absence of Cu2+. The degree of protection was found to approach maximum at about 5 mmol/l Cu2+. The well-known cariostatic properties of Cu2+ may therefore be due not only to its ability to inhibit bacterial growth but also to its ability to directly inhibit acid dissolution of enamel.

Previous freeze-etching studies of developing enamel revealed collinear arrays of spherical structures (approximately 50 nM dia) of similar width to the crystals of mature tissue. Concomitant with matrix degradation/processing, spherical structures became less distinct until, coincident with massive matrix loss, only crystal outlines were seen. More recently, using Atomic force microscopy technology, early crystals exhibited topology reminiscent of these collinear spherical structures. After matrix loss these were replaced by similarly sized bands of positive charge density on the crystal surfaces. The data suggest enamel crystals may form from mineral-matrix spherical subunits. Matrix processing may generate mineral nuclei and lead to their fusion and transformation into long apatite crystals. Support for this view derives from the appearance of short crystal segments in amelogenesis imperfecta (hypoplastic AI) or abnormally large crystals alongside 50 nM diameter spherical mineral subunits (hypomaturation AI). Mutation of matrix or processing enzymes leading to defective processing may have impaired mineral initiation, fusion, and subsequent growth.

In common with many biofilms in nature, oral plaque has been shown to have a heterogeneous structure as shown by confocal microscopy. In the present study we have used confocal laser scanning microscopy (CLSM) to determine changes in the structure of plaque biofilms with increasing plaque age. Natural human plaque biofilms were formed using the Leeds in situ device. Plaque biofilms were allowed to form for periods of 2 days, 1 week, 2 weeks and 4 weeks before removal of the devices and immediate imaging using the CLSM. Confocal imaging showed that the biofilms retained their heterogeneous appearance at each of the time points studied but appeared to have a tendency to become somewhat more dense with increasing time. Image analysis demonstrated that the percentage of biomass within each biofilm increased over time, particularly between 2 days and 1 week, and with increasing depth into the biofilms. In addition, using the CLSM we were able to demonstrate changes in the bacterial flora of the biofilms with time, with many more filamentous forms being present at the 2- and 4-week time points.

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.

The tissue compartmentalization of enamelin-processing products has been investigated in developing pig enamel using a sequential extraction procedure. Only trace amounts of enamelin-processing products were detected in simulated enamel fluid extracts, suggesting that enamelins are not solubilized in the matrix to any great extent. Subsequent phosphate buffer extraction desorbed and extracted several enamelin-processing products that were presumably bound to the mineral phase. A 35-kD processing product dominated the phosphate extract, suggesting that enamelin processing leads to an accumulation of this mineral-bound molecule. Dissociative extraction with urea subsequently extracted the remainder of the enamelin-processing products present. This material was presumably present in the tissue in an aggregated insoluble state. Several enamelin-processing products were only extracted by specific extraction procedures, suggesting that different enamelin-processing products are differentially compartmentalized. This may indicate that specific enamelin-processing products have different functions. In contrast to amelogenins, which are processed in the deeper tissue to generate products having a low affinity for the mineral, enamelin processing appears to produce products (those enamelins desorbed by phosphate buffer) that have a high affinity for the mineral. These products, appearing in the deeper enamel layers, may serve to influence crystal growth kinetics in the absence of any mineral-binding amelogenins.

Hypomaturation amelogenesis imperfecta (AI) is characterized clinically by enamel of normal thickness that is hypomineralized, mottled, and detaches easily from the underlying dentin. Autosomal dominant, autosomal recessive, X-linked, and sporadic modes of inheritance have been documented. The present study investigated the elemental composition of the enamel of teeth from individuals demonstrating clinical hypomaturation AI from families representing three of these patterns of inheritance. The aim of the study was to determine if there was any commonality in microscopic phenotype of this defect between families demonstrating the various inheritance patterns. One section from each tooth was microradiographed and then viewed in a scanning electron microscope (SEM) equipped with an ultrathin window energy-dispersive x-ray spectroscopy (EDX) detector. In the SEM, prisms and constituent crystals in discrete areas appeared to be largely obscured by an amorphous material. EDX analysis showed enamel outside these areas to have a composition indistinguishable from control teeth. However, within these affected areas there was a large increase in carbon content (up to a fivefold increase). In some teeth there was also a detectable but smaller increase in the relative amounts of nitrogen or oxygen. The results suggest the defect in these teeth with a common clinical phenotype, irrespective of the pattern of inheritance, demonstrates a commonality in microscopic phenotype. The large increase in carbon content, not matched by an equivalent increase in nitrogen or oxygen, suggests a possible increased lipid content. In those teeth with elevated nitrogen levels there may also be retained protein.

Objectives: Recently, a novel device to generate dental plaque in situ on a removable human enamel surface was described. The device permitted the recovery of plaque intact and undisturbed on its enamel substrate. The aim of this investigation was to determine the utility and robustness of this model for analysis of the effects of therapeutics on both enamel remineralisation and on the overlying biofilm composition., Methods: Enamel slices were taken from extracted sound human teeth, sterilised and a 'flat' area ground on each slice. An artificial lesion was formed within this area using acidified gel and the hardness of the enamel within the area of the lesion was determined at five sites using a 'Vickers' indenter. A nylon ring was then attached over the area of the lesion with cyanoacrylate and the excess enamel removed to form the completed device. Two devices were attached to the upper molars of 22 volunteers. Each volunteer was randomly assigned to receive either a fluoride containing (1500 ppm) or a fluoride free dentifrice. The devices were retained for a 4 week period whilst undertaking normal oral hygiene. All procedures were conducted according to GCP. After a 2 week break, the volunteers were fitted with two further devices, given dentifrice of the alternate type and the procedure repeated. Plaque from each device was harvested for microbiological analyses and the enamel subject to microhardness measurement. Then for each device the change in microhardness of the enamel within the lesion over the 4 week period was calculated., Results: There were no significant differences in viable counts of total aerobic bacteria, mutans streptococci or lactobacilli, nor in acidic, aciduric or arginolytic populations in plaque from patients using the two different dentifrices. However, devices subjected to the fluoride containing dentifrice demonstrated a significantly greater increase in microhardness of the enamel (P<0.025)., Conclusions: These results suggest that the novel in situ device is capable of measuring the effect of 1500 ppm fluoride on remineralisation of carious enamel over a 4 week period and is also well suited to determining concomitant effects on plaque ecology.

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.

Amelin (also known as ameloblastin and sheathlin) is a recently described protein that is secreted by ameloblasts during enamel formation. Here, the extracellular distribution and processing of amelin during rat incisor amelogenesis were investigated by Western blot probing using anti-recombinant rat amelin antibodies. In addition, the solubility behaviour and aggregative properties of rat amelin were investigated using a sequential extraction procedure involving (1) extraction with simulated enamel fluid to extract proteins most likely to be soluble in vivo; (2) extraction with phosphate buffer to desorb proteins bound to enamel crystal surfaces; (3) extraction with sodium dodecyl sulphate (SDS) to extract proteins present as insoluble aggregates; followed by (4) a final acid demineralization step to release any remaining proteins. Proteins immunoreactive to the anti-amelin antibodies were detectable in secretory- and transition-stage enamel. Maturation-stage enamel appeared devoid of amelin. The largest immunoreactive protein detected migrated at 68 kDa on SDS gels, corresponding to the M(r) of nascent amelin. Other immunoreactive bands at 52, 40, 37, 19, 17, 16, 15, 14 and 13 kDa were presumably amelin processing products. The sequential extraction procedure revealed that the 68-, 52-, 40-, 37- and 13-kDa amelins were completely extracted under solution conditions similar to those reported to exist in vivo. In contrast, the 19-, 17- and 16-kDa amelins were only partially extracted, whilst the 15- and 14-kDa amelins could not be extracted with simulated enamel fluid. A proportion of the remaining 17- and 16-kDa amelins was desorbed from the enamel crystals with phosphate buffer and appeared to have been mineral-bound. The 15- and 14-kDa amelins and the remainder of the 17- and 16-kDa amelins were extracted with SDS only, suggesting that these species were present in vivo as an insoluble aggregate. The results provide additional information on amelin processing and degradation, and on how such processing influences the solubility and aggregative properties of amelin-derived proteins.

Dental fluorosis is an increasing problem, yet the precise mechanism by which fluoride exerts its effects remains obscure. In the present study, we have used atomic force microscopy to image and quantitate surface features of enamel crystals isolated from specific developmental stages of fluorotic and control rat incisors. The results showed a significant decrease in crystal surface roughness with development in control tissue. Crystals from fluorotic tissue were significantly rougher than controls at all stages of development, did not decrease in roughness during the later stages of their development and had many morphological abnormalities. These data clearly demonstrate an effect for fluoride on enamel crystal surfaces which could reflect changes in the nature and distribution of growth sites and/or in mineral-matrix interactions. These would be expected to affect crystal growth during maturation, resulting in the characteristic porous appearance of fluorotic lesions in mature teeth.

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.

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was used to investigate the subunit structure of the developing enamel matrix. Intact matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subunit composition determined by analytical SDS-PAGE following reductive cleavage of the cross-links. Western blotting using antiamelogenin antibodies was used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelogenins while amelogenin-processing products tended to be cross-linked to other processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and during extracellular processing, and that processed products are not free to associate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules within supramolecular aggregates (nanospheres) are processed in situ and remain in the same nanosphere while all the individual component amelogenins undergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin-amelogenin associations are maintained during processing indicates that matrix stability is an important factor while the enamel layer is being deposited.

Background: The aim of the present study was to compare the morphology and elemental composition of subgingival calculus between Indo-Pakistani and Caucasian patient groups., Methods: Extracted teeth from 14 Indo-Pakistani and 19 Caucasian subjects were collected. Of these, 12 Indo-Pakistani and 10 Caucasian teeth had sufficient subgingival calculus for analysis. Subgingival calculus present on the 22 teeth was classified into six morphological types: 1) crusty/spiny/nodular; 2) ledge/ ring; 3) thin, smooth veneers; 4) finger/fern-like; 5) individual islands/spots; or 6) supramarginal on submarginal. Subgingival calculus was zoned: coronal, mid, and apical. A sample obtained from each zone was subdivided to allow 3 separate analyses: transmission electron microscopic (TEM) x-ray microanalysis for elemental composition, fluoride analysis, and carbonate analysis., Results: Crusty/spiny/nodular, ledge/ring, and thin, smooth veneers were more commonly found in the Indo-Pakistani group; individual islands were more prevalent in the Caucasian subjects. Supramarginal on submarginal calculus was found only in the Indo-Pakistani group. No finger/fern-like deposits were found. No differences within or between the two ethnic groups were found with regard to calcium:phosphate ratios, fluoride, or carbonate content. However, the Indo-Pakistani group showed significantly lower levels of sodium in apical samples than in coronal samples (ANOVA, F1,16 = 5.98, P= 0.03), and significantly lower levels of sodium (ANOVA, F1,12 = 4.75, P= 0.05) and magnesium (ANOVA, F1,12 = 5.16, P= 0.04) in apical samples than in those from Caucasians. After adjusting for smoking, the magnesium results remained significant (ANOVA, F2,11 = 4.64, P= 0.05)., Conclusions: Subgingival calculus demonstrated differences in morphology between these two ethnic groups and differences in elemental composition, which may influence its overall solubility and contribute to its greater accretion in the Indo-Pakistani subjects.

We recently developed a simple and inexpensive method that complements established bone histomorphometry procedures by enabling the two-dimensional imaging of cancellous bone to be viewed within its three-dimensional context with the marrow tissue in place and without detriment to the material for other histological purposes. The method, based on the preparation and superficial staining of slices 300 microm thick, enables "real" (i.e., unstained) trabecular termini to be separated from "artifactual" (i.e., stained) termini, providing a direct measure of cancellous connectivity in osteopenic bone. The technique was applied to osteopenic age-matched, white, postmenopausal women (31 with and 22 without vertebral compression fractures) with a similar bone status, as measured at the spine by absorptiometry and at the iliac crest by histology (see part I of this study). Despite the similarity in the mass of trabecular bone at either site, the results showed a significant difference (p < 0. 05) in the number of "real" trabecular termini between the groups, such that the fracture group had almost four times as many termini (mean +/- SE: 1.98 +/- 0.51/30 mm(2)) at the iliac crest as the nonfracture group (mean +/- SE: 0.53 +/- 0.31/30 mm(2)). Previous histomorphometry of the same material failed to detect a structural distinction between the two groups using established variables. It was concluded that a mass-independent trabecular discontinuity contributes to skeletal failure and that determination of the number of "real" disconnections (i.e., unstained termini) by the direct method proposed may provide a more sensitive discriminant of fracture than the present indirect procedures. A group of fracture and nonfracture men (see part I) suggested a similar distinction (fracture: 0.69 +/- 0.30/30 mm(2); nonfracture: 0.18 +/- 0.18/30 mm(2)), although the difference was not significant.

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 microm thick slices of methylmethacrylate embedded material rather than the more usual thin 8 microm histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, "real" (i. e., unstained) trabecular termini can be distinguished from "apparent" (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphometry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.

The junctionalis form of epidermolysis bullosa (EBJ) is associated with a number of clinical problems involving tooth enamel, including increased susceptibility to caries. The aim here was to carry out a chemical characterization of the enamel of teeth from EBJ patients compared with that of unaffected controls. The results showed that while protein concentration, amino acid composition and carbonate content were similar in both groups, EBJ enamel contained a significantly reduced mineral per volume content, resulting in enamel hypoplasia. In addition, Western blotting revealed the presence of serum albumin (a known inhibitor of enamel crystal growth) in EBJ enamel. This was not detected in control enamel or in enamel of teeth from patients with the dystrophic form of the disease. It is concluded that EBJ enamel is developmentally compromised and that the enamel defects are commensurate with the reported genetic lesions.

While it is believed that proteins may protect enamel from demineralisation, recent work has indicated that such material may also hinder remineralisation. For example, albumin will inhibit apatite crystal growth in vitro and is present in carious enamel in vivo. However, it is not clear whether (1) the distribution of proteins within lesions is restricted to specific lesion zones or (2) the origin of such proteins is endogenous (i.e. as a remnant of the developmental process) or exogenous, originating in the saliva or gingival crevicular fluid. The present study used a combination of immunohistochemistry and microradiography to determine the distribution of two proteins, serum albumin and salivary amylase, within natural white-spot carious lesions in relation to specific levels of demineralisation. The results indicated that albumin is found primarily in a region of between 10 and 20% demineralisation (an area of transition between the 'dark' zone and lesion 'body'), with smaller quantities occurring in the region between 0 and 10% demineralisation and trace amounts in the zone indistinguishable from sound enamel by microradiography. A similar distribution was found for amylase in that the heaviest labelling was within the 10-20% demineralisation zone, although little if any was present in the 0-10% zone. The presence of these molecules in a region of the lesion where some potential for reprecipitation may exist will have important implications with respect to lesion progression.

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 microm) than in the center of the devices (range = 35-215 microm). 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.

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.

Carious attack on enamel is not a unidirectional process but involves both demineralisation and remineralisation. The chemistry of carious attack on enamel has, to a large extent, now been clarified as far as mineral components are concerned but little attention, however, has been paid to the identity of organic material in carious lesions and its possible role in the caries process. The only clear information available is that organic material accumulates with time within enamel lesions. The present study was aimed at identifying a specific protein component known to bind to hydroxyapatite (albumin) in carious lesions with a view to investigating its role in the disease process. The distribution of albumin within both white spot and fissure lesions and adjacent sound enamel of extracted human teeth was investigated using SEM immunohistochemistry on undermineralised sections of human enamel and employing a polyclonal antibody to human serum albumin. The nature of the protein, i.e. whether it was in the form of intact molecules or degraded fragments, was investigated by Western blotting, employing the same antibody. The immunohistochemistry revealed the presence of albumin within both interproximal white spot and fissure lesions with little if any present in sound enamel. The Western blotting indicated that the albumin was in the intact form with no evidence of degradation products. The ability of albumin to bind and to inhibit growth of calcium phosphate crystals raises the question as to the possible role of such a molecule in the development of carious lesions.

During the secretory stage of enamel development, the hydroxyapatite crystals appear as thin ribbons which grow substantially in width and thickness during the later maturation stage. In this study, the atomic force microscope (AFM) was used to investigate developmentally-related changes in deproteinized enamel crystal surface topography in normal animals and in those receiving daily doses of fluoride. The AFM revealed previously undescribed surfaces features, some of which may represent growth sites or different crystalline phases. Secretory stage crystals had greater surface rugosity and were more irregular, with spherical sub-structures of 20-30 nm diameter arranged along the "c"-axis. Maturation stage crystals were smoother and larger but revealed both subnanometer steps and lateral grooves running parallel to the "c"-axis. Crystals from fluorotic tissue showed similar features but were more irregular with a higher degree of surface roughness, suggesting abnormal growth. The AFM may prove an important adjunct in determination of the mechanisms controlling crystal size and morphology in skeletal tissues.

The hydroxyapatite crystals of mature enamel are unusually large, uniform and regularly disposed within the tissue, implying that their development is a highly controlled process. The organic matrix of developing enamel is presumed to play an important role in the modulation of mineral deposition and growth during tooth morphogenesis but the precise functions of individual matrix proteins remain unclear. The aim of this review was to survey the current knowledge of enamel matrix proteins with a view to suggesting possible functions. The organic matrix is highly heterogeneous, comprising proteins derived from a number of different genes, including amelogenin, enamelin, ameloblastin (amelin/sheathlin), tuftelin, dentine sialophosphoprotein, enzymes and serum proteins such as albumin. Each of these classes appears to undergo post-secretory sequential degradation which contributes further towards matrix heterogeneity. Possible functions of these proteins include de novo mineral nucleation/initiation (dentine sialophosphoprotein, tuftelin), mineral ion binding as crystal precursors (amelogenin, enamelin), control of crystal growth (amelogenin, enamelin, ameloblastin), support of growing crystals (amelogenin, enamelin), determination of prismatic structure (ameloblastin), cell signalling (tuftelin, ameloblastin), control of secretion (breakdown products) and protection of the mineral phase (amelogenin, enamelin). Failure of these mechanisms could lead to incomplete maturation of the enamel and the eruption of dysplastic tissue.

Enamel formation is a complex process which involves the expression of a number of genes, the most obvious being those related to the mineralized extracellular matrix. In this study the differential-display technique, first described by Liang and Pardee, has been used to identify genes specifically expressed in enamel organ cells. By comparing results obtained from RNA derived from rat enamel organ with RNA derived from other cellular sources, a number of differentially expressed transcripts have been identified. The nucleotide sequences of two of these have been analyzed and shown to have no homology with any previously published sequences. Further analysis will provide information on the type of protein that they may encode, their tissue distribution and their potential role in enamel formation.

Processing of enamel matrix proteins is essentially biphasic. Secretory stage metalloprotease activity generates a discrete, presumably functional, spectrum of molecules which may also undergo dephosphorylation. Maturation stage serine proteases almost completely destroy the matrix. The present aim was to examine the tissue compartmentalization of these enzyme activities in relation to their possible function. A sequential extraction using synthetic enamel fluid, phosphate buffer and SDS was used to identify enzymes free in the enamel fluid, crystal bound or aggregated with the bulk matrix respectively. Results indicated that the metallo-proteases and alkaline phosphatase were free in the secretory stage enamel fluid while the serine proteases appeared to be largely bound to the maturation stage crystals. The mobility of the metallo-proteases and alkaline phosphatase would ensure efficient initial processing of secretory matrix, while the largely mineral bound serine proteases would ensure retention of protease activity despite massive destruction and protein removal.