Oral Cells and Tissues. Philias R. Garant
Ameloblastin degradation products are less soluble than the parent molecule.43 Nonamelogenin protein fragments are believed to account for most of the remaining small percentage of protein contained in fully mature enamel.
Of additional interest is the report that amelin mRNA has been localized in preodontoblasts before it is expressed in ameloblasts.78 It has been suggested that amelin may have a role in the presecretory epithelial-mesenchymal interaction.
Enamelin is a high–molecular weight, acidic, glycosylated protein. It is secreted as a 186-kDa entity that subsequently undergoes progressive degradation to a 32-kDa molecule.81 The later fragment binds readily to enamel crystallites.82 The higher molecular weight fractions are localized along Tomes process and the newly developing enamel prism. The smaller fractions are located more deeply in the enamel, in association with the mineral in the prismatic and interprismatic domains.81,83
Some acidic proteins of the enamelin fraction are now known to be precursors of a group of serine proteinases that degrade enamel matrix.58,68,69,84
Despite recent progress in the biochemical characterization of the enamel matrix, knowledge of the sequential biochemical and biophysical interactions between mineral ions and enamel matrix proteins is still very incomplete. The most recently postulated roles for amelogenin and nonamelogenin proteins in initiating and controlling the construction of enamel have been reviewed by Nanci et al,16 Robinson et al,43 and Fincham et al.51
Location and Expression of Amelogenin, Ameloblastin, and Tuftelin Genes
The gene for amelogenin (AMEL) has been mapped to the sex chromosomes (Fig 3-7). In the rat, hamster, and mouse, Amel is present on the X chromosome85; in humans, AMEL is present on both the X and Y chromosomes.86,87 The gene on the Y chromosome (AMELY) is located in the q11 region, and the AMELX gene is located on the distal short arm (p22.1 to p22.3 positions) of the X chromosome. Recombination errors during the duplication of the sex chromosomes can lead to amelogenesis imperfecta.
Fig 3-7 Structure of the X-chromosomal copy of the human amelogenin gene. The bar segments represent the introns and the boxes (1 through 7) correspond to the exons. The nucleotide numbers are indicated below the exons. (Adapted from Simmer et al.173)
The human amelogenin gene has seven introns and seven exons (see Fig 3-7). Both the X and Y amelogenin gene copies are expressed during tooth development. Transcription of the AMELX message appears several times more active than that of the Y copy, and the level of X-chromosomal amelogenin mRNA has been measured to be several fold higher than that of Y-chromosomal amelogenin mRNA.
A variety of amelogenin proteins are produced by alternative splicing of pre-mRNA.46–48 Exons, and parts of exons, are deleted during alternative splicing. The resulting proteins all have a hydrophobic amino terminal, a large hydrophilic middle polypeptide, and a hydrophilic carboxy terminal. It is unclear if each amelogenin isoform performs a different function during enamel formation.
A small deletion in the AMELY gene permits it to be distinguished from its AMELX counterpart. This difference has proven useful in sex identification of human remains recovered from archeological sites88 and in forensic science.89
The human tuftelin gene is located on chromosome 1.74,90 The ameloblastin gene is localized to chromosome 4q21.91
Mineralization of the Enamel Matrix
At the onset of enamel formation, the first enamel crystallites are spatially separated from the smaller dentin crystallites. High-resolution electron microscopy of the dentinoenamel junction indicates that the earliest enamel crystallites form from the alignment of dotlike mineral nuclei, approximately 2 to 4 nm in diameter.92 Chainlike association of these nuclei, apparently controlled by the amelogenin organic matrix, gives rise to long, needle-shaped crystallites. The crystallites develop in small clusters within extracellular deposits of amelogenin matrix, having the appearance of stippled material in electron micrographs.
Biochemical and electron probe analysis of the earliest crystallites suggests that the first mineral phase to be formed is a two-dimensional octacalciumphosphate precursor that subsequently transforms into hydroxyapatite.66,93 The smallest hydroxyapatite crystal units (unit cells) are formed by the following reaction:
The hydrogen ions generated during crystal formation must be buffered to maintain a neutral pH to allow continued matrix mineralization.44
Enamel crystal growth occurs in a compartment isolated between mineralized dentin and the zonula occludens junction of the ameloblastic layer. Elemental analysis indicates that the fluid in the mineralization compartment has a different composition than serum and extracellular fluid.94,95 The presence of a distal zonula occludens junction between ameloblasts and the histochemical demonstration of calcium ATPase activity in the plasma membrane of Tomes process suggest that ameloblasts might control the fluid milieu within which enamel is deposited.96–98
Calcium ATPase has also been demonstrated in the distal cytoplasm of maturation ameloblasts.98 The recent localization of Ca2+ pump proteins in human secretory and early-stage maturation ameloblasts provides additional support for a functional plasma membrane calcium pump.99 The highest concentration of calcium pump protein was localized in the distal ends of ameloblasts near the mineralized enamel.99
It has been proposed that the intracellular transport of calcium could be carried out by several calcium-binding proteins localized in both secretory and maturation ameloblasts.100–102 The recent identification of two low-affinity, high-capacity, calcium-binding proteins (calreticulin and endoplasmin) in the endoplasmic reticulum of secretory and maturation ameloblasts provides support for a new theory of calcium transcytosis involving the endoplasmic reticulum and inositol triphosphate–gated calcium channels.103 The endoplasmic reticulum could serve as a high-volume conduit for calcium transport across ameloblasts without altering the normal cytosolic calcium concentration. This theory would also explain why a large amount of endoplasmic reticulum but low levels of secretory protein synthesis are found in maturation ameloblasts.103,104
In situ hybridization with complementary deoxyribonucleic acid (cDNA) probes for bone sialoprotein is strongly positive in secretory ameloblasts. The potential role of bone sialoprotein, a calcium-binding protein common to most mineralized tissues, in enamel mineralization remains to be determined.105 It has been suggested that tuftelin and/or bone sialoprotein could trigger enamel crystal nucleation.43,73
Structure of Transition-Stage Ameloblasts
On completion of the full thickness of enamel, the secretory ameloblasts undergo cytoplasmic reorganization as they switch from a primarily protein secretory cell to that of an absorptive and transport cell. This process is characterized by extensive intracellular digestion of parts of the RER and other cytoplasmic organelles inside autophagosomes. During this stage, the ameloblasts contain high levels of acid phosphatase, indicative of increased lysosomal enzyme activity. The transition stage remodeling is so intensive that approximately 25% of the ameloblasts undergo programmed cell death.106,107