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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.

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.

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.

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 effect of removal of glycosylaminoglycans on the mineralization of sheep periodontal ligament was determined using enzyme digests followed by incubation in solutions supersaturated with respect to hydroxyapatite at pH 7.4. TEM revealed that control periodontal ligament remained unmineralized. However, tissue from which glycosylaminoglycans had been removed contained plate-like crystals arranged parallel to and within the collagen fibrils. Electron probe and electron diffraction studies suggested that the crystals were apatitic with a similar order of crystallinity to dentine, and a Ca:P ratio of 1.61. In addition, the glycosylaminoglycan content of periodontal ligament, cementum and alveolar bone was compared using cellulose acetate electrophoresis. Periodontal ligament contained predominantly dermatan sulfate while cementum and alveolar bone contained mostly chondroitin sulfate. A role for glycosylaminoglycans in maintaining the unmineralized state of the periodontal ligament is suggested. Control of expression of specific proteoglycan species on a spatially restricted basis is presumably central to this role.