While the participation of the extracellular matrix protein in mineralisation processes has been extensively studied, a role for these proteins as signalling molecules has only been recently evoked. We report on the composition of enamel and dentin matrix, and discuss the multifunctionality of two molecules. Amelogenins are implicated in enamel three-dimensional organization and contribute to the initial mineralisation of the forming enamel. Some amelogenin spliced forms act as signalling molecules in odontoblasts. Dentin Matrix Protein-1 is also implicated in dentin mineralisation and in the differentiation of pulp embryonic cells into odontoblasts. This reveals a complex role of extracellular matrix molecules.
The view that the molecules of the extracellular matrix are only implicated in the mineralisation of calcified tissues now appear simplistic in view of the number of articles also reporting signalling functions to some of these components. It is nowadays well recognized that the cells synthesize intracellular, pericellular and a complex extracellular matrix (ECM), especially dense and important in mineralised tissues. ECM molecules act as a three-dimensional template to promote nucleation, followed by crystal growth. ECM molecules also provide appropriate volume within which the mineral is formed. They are organized as complex hydrophobic spaces defined by glyco-lipo-proteinic ‘envelopes' (crystal ghosts in bone and dentin, matrix vesicles in bone and cartilage, tubules or nanospheres in enamel), which allow the accumulation of calcium and phosphate ions. These calcium and phosphate ions originate from the blood or interstitial fluids bypassing leaky junctional complexes together with their carrier molecules, such as albumin or lipids. A cascade of mineralisation reactions driven by thermodynamically unstable steps leads to the stabilization of the amorphous calcium phosphate, which is first transformed into calcium triphosphate, and then into calcium octophosphate. This forming mineral phase is further transformed into a thermodynamically more stable hydroxyapatite (HAP) form of lower energy level. After having induced the nucleation of the first calcium phosphate crystals, ECM components are then implicated in the control of crystal growth
The global composition of enamel and dentin indicates substantial differences in composition of the two structures, which may interfere with their final biological and biomechanical properties (
Data related to the enamel matrix composition are summarized in
The composition of dentin extracellular matrix proteins is similar to that of bone and cementum. However, proteins such as dentin sialoprotein (DSP), and also the dentin phosphoprotein (DPP), present both in dentin and bone, are much more abundant in the dentin (approximately 400 times). Other proteins are mostly expressed in bone (bone sialoprotein, osteopontin), but are also found in dentin, at least at early stages of tooth formation. In contrast, other proteins, such as DMP-1, are present in equal amount in both bone and dentin.
Dual or multipotent biological properties of some of these molecules will be examined in the next parts of this review. It is obvious that these molecules are implicated in the formation of the organic scaffold and consequently in the mineralisation processes. However, it becomes clear nowadays that they are also involved in signalling functions of the cells that are responsible for their synthesis: the secretory odontoblasts and ameloblasts.
Amelogenins are expressed by ameloblasts during enamel formation. A heterogeneous mixture of amelogenins has been extracted from the forming enamel with large variations in MW from 28kDa to 5kDa. These multiple forms have been attributed to alternative splicing
Three domains have been identified in the amelogenin molecule. The first is a highly conserved amino-terminal ‘TRAP’ (tyrosine-rich amelogenin peptide) sequence of 44/45 residues. This sequence is phosphorylated in serine-16, but not glycosylated. The TRAP segment has lectin-like properties and is similar to wheat germ agglutinin as it binds
Although amelogenins do not possess in vitro the specific crystal-modulating properties characteristic of certain acidic mineralised tissue proteins the supra-molecular control of HAP crystal growth is under the control of amelogenin amelogenin-deficient mice display an amelogenesis imperfecta phenotype and disorganized hypoplastic enamel (this suggests that amelogenins are not required for the initiation of mineral crystal formation, but rather for the organization of crystal pattern and for the regulation of enamel thickness finally, N-terminal-deleted amelogenin has been seen to induce in vivo severe enamel defects in mouse, whereas C-terminal-deleted amelogenin does not alter enamel formation
The importance of amelogenins in enamel formation has been clearly demonstrated. The molecules are already expressed by presecretory ameloblasts, but no enamel is formed at the early stages, as the secreted enamel proteins presumably diffuse through the porous predentin and the unmineralized mantle dentin. At least some of these proteins, and in particular the different low-molecular-weight species of amelogenin are thought to be internalised into odontoblasts, thus preventing their accumulation in the dentin. The internalised amelogenins are then presumably destroyed in the lysosomes of the odontoblasts. Amelogenin diffusion is gradually diminished by dentin mineralisation, causing amelogenins and the other enamel proteins, to be retained at the dentin–enamel junction (DEJ), initiating enamel formation.
Two series of data have demonstrated that the situation is in fact more complex and suggest that the amelogenin peptides may actually serve as signalling molecule in the secretory odontoblasts. Firstly, the demonstration of amelogenin splice products in the odontoblast gene library
The group of Veis
Taken together, these data suggest that amelogenins are not only involved in enamel formation, but may also function as signalling molecules. This property of amelogenin is currently exploited therapeutically in periodontal and bone regeneration (Emdogain). Our experiments open gates on the healing of pulp damages.
During crown formation, the enamel organ plays a role in regulating enamel formation and consequently in the secretion and diffusion of amelogenins in dentin. During root formation, the epithelial Hertwig's root sheath is instrumental in the recruitment and differentiation of root odontoblasts. For a long time, differences between the root and crown dentin have been recognized. It is also the case for the coronal and root pulp
Our recent immunohistochemical and immunogold electron microscopic results confirmed the presence of MMP-20 protein in both ameloblasts and odontoblasts of rat incisor and showed that MMP-20 protein colocalizes with amelogenin, suggesting that in odontoblasts too, amelogenin may be the target substrate. This co-localization of amelogenin and MMP-20 would argue for local synthesis of both enzyme and substrate.
Unlike ameloblasts, where MMP-20 is found to be expressed in the cells and secreted into the matrix, suggesting a role in the processing of enamel proteins leading to enamel maturation, MMP-20 expressed in odontoblasts was not detected in the corresponding predentin/dentin matrix. This suggests that the local degradation of amelogenin by MMP-20 in odontoblasts would produce low-molecular-mass peptides, which may serve as signalling molecules. A schematic representation of the possible various pathways and hypothesis is shown in
The distribution of dentin sialoprotein (DSP) or the staining of dentin phosphoprotein (DPP) with the ‘stains all' or with the phosphotungstic acid/chromic acid, both revealing phosphorylated proteins, stained the predentin–dentin junction but not the predentin. These observations provided evidence that these two daughter molecules resulting from the dissociation of the native DSPP are involved in dentin mineralisation. Autoradiographic data by Weinstock and Leblond
In contrast, the immunolocalization of dentin matrix protein-1 (DMP-1), another phosphorylated protein member of the SIBLING family, was positive in odontoblasts and, when the staining was missing in odontoblasts, was shifted to presecretory and secretory ameloblasts (
To conclude, the two examples given here provide evidence of the dual functions of the molecules of the extracellular matrix, which now appear more complex than previously thought. The multifunctionality of extracellular matrix molecules is now under investigation in many laboratories.
Alteration of the apposition zone of the forming enamel of a N-terminal-deleted amelogenin mouse. Stippled material (SM) is abundant. Electron-dense apoptotic nuclei are seen.
Fig. 2. Structural alterations of the forming enamel of a N-terminal deleted mouse. DEJ: dentino-enamel junction.
Fig. 1. Altération de la zone d'apposition de l'émail en formation chez une souris à amélogénine ayant une délétion N-terminale. Le matériel punctiforme (SM) est abondant. On observe des noyaux en apoptose denses aux électrons.
Fig. 2. Altération structurale de l'émail en formation chez une souris portant une délétion N-terminale. DEJ = fonction amélo-dentinaire.
Schematic representation of ameloblastic and odontoblastic amelogenins, pathways of degradation and signalisation.
Fig. 3. Représentation schématique des voies de dégradation et de signalisation des amélogénines amélo- et odontoblastiques.
Immunohistochemical staining of DSP. The odontoblasts and mineralisation front are positively stained.
Fig. 5. Anti DMP-1 staining. Positive reaction is seen in odontoblasts and in a few enamel organ cells.
Fig. 6. After inhibition of phosphorylation by inositol hexasulphate, immunostaining for DMP-1 is seen in ameloblasts and in the enamel organ.
Fig. 4. Coloration immunohistochimique de sialoprotéine dentinaire (DSP). Les odontoblastes et le front de minéralisation sont positivement colorés.
Fig. 5. Coloration dirigée contre le DMP-1. Une réaction positive s'observe dans les odontoblastes et dans quelques cellules de l'organe de l'émail.
Fig. 6. Après inhibition de la phosphorylation par l'hexasulfate d'inositol, l'immunocoloration pour la DMP-1 s'observe dans les améloblastes et dans l'organe de l'émail.
Global composition in weight and volume of two dental tissues: enamel and dentin
Composition globale en poids et volume de deux tissus dentaires : émail et dentine
Enamel matrix composition
Composition de la matrice de l'émail
Dentin matrix composition
Composition de la matrice de la dentine