Hypomaturation amelogenesis imperfecta due to WDR72 mutations: a novel mutation and ultrastructural analyses of deciduous teeth.

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.

Structural changes in the ageing periosteum using collagen III immuno-staining and chromium labelling as indicators.

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.

Ultrastructural analyses of deciduous teeth affected by hypocalcified amelogenesis imperfecta from a family with a novel Y458X FAM83H nonsense mutation.

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.

The structure and composition of deciduous enamel affected by local hypoplastic autosomal dominant amelogenesis imperfecta resulting from an ENAM mutation.

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.

Self-assembling peptide scaffolds promote enamel remineralization.

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.

Plaque biofilms: the effect of chemical environment on natural human plaque biofilm architecture.

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.

Preservation of thoracic spine microarchitecture by alendronate: comparison of histology and microCT.

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.

Penetration of fluoride into natural plaque biofilms.

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

Surface chemistry of enamel apatite during maturation in relation to pH: implications for protein removal and crystal growth.

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.

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.

Minimizing prion risk without compromising the microbial composition of biofilms grown in vivo in a human plaque model.

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.

Subunit structures in hydroxyapatite crystal development in enamel: implications for amelogenesis imperfecta.

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.

Copper ions inhibit the demineralisation of human enamel.

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.

Inheritance pattern and elemental composition of enamel affected by hypomaturation amelogenesis imperfecta.

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.

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.

Enamelin compartmentalization in developing porcine enamel.

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.

Changes in the structure and density of oral plaque biofilms with increasing plaque age.

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.

Investigation to evaluate and validate the Leeds in situ device for the study of enamel remineralisation in vivo.

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.

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.

Amelin extracellular processing and aggregation during rat incisor amelogenesis.

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.