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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.) more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

A monoclonal antiamelogenin antibody was used to investigate the effects of fluoride on enamel development in the rat incisor. The results suggested that during secretion the enamel matrix molecules are arranged in such a way as to mask the epitope recognized by the monoclonal antibody. However, during the transition stage of development as the matrix begins to be degraded the epitope becomes exposed and labelling intensity increases to reach a maximum at the end of transition/start of maturation. The effect of fluoride is to delay the appearance of labelling within the enamel matrix until the end of transition. This suggests that the fluoride may inhibit enzymatic degradation or disaggregation of the matrix, the resulting residual matrix then inhibiting crystal growth. more...

The effect of accelerated odontogenesis of the rat mandibular incisor on the expression of receptors for epidermal growth factor (EGF) was examined using specific monoclonal antibodies to the receptor molecule. Acceleration of odontogenesis was achieved by regular trimming of the tooth crown. At normal eruption rates the major area of cross-reactivity was over the secretory ameloblasts. Some labelling of the papilla and preameloblasts was evident. When proliferation was increased the major area of effect was at the preodontoblast/odontoblast boundary where there was a marked increase in labelling, initially at the proximal end of the cell adjacent to the basal lamina. The ameloblasts did not show such a dramatic increase in receptor numbers. Increase in labelling was also evident in the remainder of the papilla. The results suggest that an increase in proliferation with normal morphogenesis is associated with an overall increase in the numbers of EGF receptors, particularly in a population of cells immediately before and after elongation and differentiation of odontoblasts. more...

A 0.15 per cent solution of aminoacetonitrile was added to the drinking water of young adult male Wistar rats for 18 days. Their right mandibular incisors were maintained unimpeded by frequent trimming. After 18 days, the periodontal ligaments of the right mandibular incisors and first molars were prepared for light and electron microscopy. For control purposes, similar material was obtained from animals fed on a normal diet ad libitum or pair-fed. With light microscopy, lathyritic incisor periodontal ligaments showed areas of normal connective tissue appearance interspersed with areas of degeneration. In the lathyritic molars, cell-free areas were observed. With electron microscopy, quantification of features in the connective tissue regions of the lathyritic ligament showed little change. In cell-free areas, many collagen fibrils had smaller fibril diameters than normal. more...

Quantitative ultrastructural studies were conducted on eight rats (four aged 21 days and four aged 8 weeks) using the periodontal tissues around the mesial root of the mandibular first molar. The periodontal ligament of the erupting tooth contained significantly more capillary fenestrations than the erupted tooth, both in terms of the number per unit area of endothelium (2.2/microns2) and in terms of total number per cubic millimetre of tissue (30.5 X 10(6)/mm3). Differences were also discerned with respect to the percentage of capillaries in the tissue. Thus, the periodontal vasculature demonstrates marked morphological changes which may be related to the eruptive phase of the tooth. more...

A quantitative, ultrastructural study was made on the periodontal ligaments of rat mandibular incisors immobilized for 18 days. Fibroblasts and their organelles (i.e. endoplasmic reticulum, mitochondria, microtubules, microfilament bundles, lysosomes, intracellular collagen profiles and intercellular contacts), oxytalan fibres, collagen fibrils and ground substance were quantified. There was re-orientation of tissue at the lateral borders of the ligament and an apparent increase in size of epithelial cell rests. Within the matrix, the mean collagen-fibril diameter decreased and the amount of ground substance increased. No change occurred in oxytalan fibres. Within the cells, effects were seen only in cell contacts and in the relative volume of intracellular collagen, lending no support to the view that fibroblast activity generates the force responsible for tooth eruption. more...

The periodontal ligaments of impeded and unimpeded rat mandibular incisors were examined to find structural correlates for the known functional differences between the tissues. The structures quantified were fibroblasts (area and membrane length, endoplasmic reticulum, mitochondria, microtubules, lysosomes, intracellular collagen profiles, intercellular contacts), oxytalan fibres, collagen fibrils and ground substance. The only changes seen on rendering a tooth unimpeded were an increase in the number of microtubules within the fibroblasts, an increase in the number of simplified desmosomes between the fibroblasts and a decreased amount of ground substance within the extracellular matrix. The results show that it is possible for a connective tissue to undergo marked changes in function, turnover and biomechanical properties without major structural changes. more...