Ectopic expression of dentin sialoprotein during amelogenesis hardens bulk enamel

Dentin sialophosphpoprotein (Dspp) is transiently expressed in the early stage of secretory ameloblasts. The secretion of ameloblast-derived Dspp is short-lived, correlates to the establishment of the dentinoenamel junction (DEJ), and is consistent with Dspp having a role in producing the specialized first-formed harder enamel adjacent to the DEJ. Crack diffusion by branching and dissipation within this specialized first-formed enamel close to the DEJ prevents catastrophic interfacial damage and tooth failure. Once Dspp is secreted, it is subjected to proteolytic cleavage that results in two distinct proteins referred to as dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). The purpose of this study was to investigate the biological and mechanical contribution of Dsp and Dpp to enamel formation. Transgenic mice were engineered to overexpress either Dsp or Dpp in their enamel organs. The mechanical properties (hardness and toughness) of the mature enamel of transgenic mice were compared with genetically matched and age-matched nontransgenic animals. Dsp and Dpp contributions to enamel formation greatly differed. The inclusion of Dsp in bulk enamel significantly and uniformly increased enamel hardness (20%), whereas the inclusion of Dpp weakened the bulk enamel. Thus, Dsp appears to make a unique contribution to the physical properties of the DEJ. Dsp transgenic animals have been engineered with superior enamel mechanical properties.     

Spatial and temporal activity of the dentin sialophosphoprotein gene promoter: differential regulation in odontoblasts and ameloblasts

Dentin sialoprotein and dentin phosphoprotein are non-collagenous proteins that are cleavage products of dentin sialophosphoprotein (DSPP). Although these two protein products are believed to have a crucial role in the process of tooth mineralization, their precise biological functions and the molecular mechanisms of gene regulation are not clearly understood. To understand such functions, we have developed a transgenic mouse model expressing a reporter gene (lacZ) under the control of approximately 6 kb upstream sequences of Dspp. The transgenic fusion protein was designed to reside within the cells to facilitate the precise identification of cell type and developmental stages at which the Dspp-lacZ gene is expressed. The results presented in this report demonstrate: (a) the 6 kb upstream sequences of Dspp have the necessary regulatory elements to direct the tissue specific expression of the transgene similar to endogenous Dspp, (b) both odontoblasts and ameloblasts exhibit transgene expression in a differentiation dependent manner, and (c) a differential regulation of the transgene in odontoblasts and ameloblasts occurs during tooth development and mineralization.     

A scanning electron microscope study of aberrations in the prism pattern of rat incisor inner enamel.

The prism pattern in the inner enamel of adult rat incisors was studied with the SEM in unfixed tissues that had been sectioned, ground, polished, and etched. Six different types of aberrations in the prism pattern were encountered: 1. Prism lamellae may be shorter than the mesio-lateral width of enamel. 2. Prism lamellae may deviate from a transverse orientation. 3. Prism lamellae may "fuse" or "bifurcate." 4. Prisms of two adjacent lamellae may pursue a common course. 5. Prisms may change direction. 6. Variations exist in the outline of transversely cut prism profiles. Aberrations were observed at any distance from the dentino-enamel junction. These observations were used as a basis for an analysis of the movement of ameloblasts during rat incisor amelogenesis. It was concluded that it is physically possible for the ameloblasts to create the observed aberrations as they move along the path of the prisms. However, the aberrations seem to make it more difficult to understand the factors controlling ameloblast movement. Occasionally crystallite bridges connecting adjacent prisms were observed. A configuration resembling a bifurcating prism is pesented.     

Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.     

The presence and possible functions of the matrix metalloproteinase collagenase activator protein in developing enamel matrix.

The developing enamel matrix contains mostly amelogenins, which are hydrophobic proline-rich proteins. During amelogenesis, the amelogenins are presumably hydrolysed and removed from the enamel. Recently a number of metalloproteinases that may be important in amelogenesis have been identified in zymograms of the developing enamel matrix. In the present study an antibody specific for the matrix metalloproteinase collagenase activator protein (CAP) was characterized and used to identify this metalloproteinase in enamel. Immunoblotting showed that the CAP proteinase was present in the enamel matrix. Immunohistochemistry confirmed that the proteinase is localized in the enamel matrix, most specifically along the dentino-enamel junction. Purified CAP was found to hydrolyse amelogenin protein. Possible functions of the proteinase in the enamel matrix are discussed.     

The Functional Width of the Dentino-Enamel Junction Determined by AFM-Based Nanoscratching

The dentino-enamel junction (DEJ) constitutes a structurally unique interphase uniting two mineralized tissues with very different matrix composition and physical properties. Its excellent biomechanical properties have drawn interest as a biomimetic model for joining dissimilar materials. In order to characterize the functional width of the DEJ, nanoscratching experiments were performed on human third molars. Friction coefficients of enamel, of dentin, and at the DEJ were obtained with a nanoscratch tester attached to an atomic force microscope (AFM). Normal loads in the range of 50 to 600 microN were applied to a spherical diamond indenter (r = 10 microm), which was driven 10 microm across the sample surface, recording the lateral force. Imaging with an AFM facilitated exact positioning of the scratches. The friction coefficient of intertubular dentin was 0.31 +/- 0.05, significantly above the coefficient of enamel of 0.14 +/- 0.02. The increased friction of dentin is attributed to the higher content of organic phases. Scratches performed across the interphase between enamel and dentin showed a sharp monotonic change in the friction coefficient. The average width of the slope between the friction coefficients of dentin and enamel was 2.0 +/- 1.1 microm and is assumed to represent the functional width of the dentino-enamel junction. The effect of the scalloped structure of the DEJ on its functional width as determined by mechanical testing is discussed.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Constitutive activation of β-catenin in ameloblasts leads to incisor enamel hypomineralization

Enamel is the hardest tissue with the highest degree of mineralization protecting the dental pulp from injury in vertebrates. The ameloblasts differentiated from ectoderm-derived epithelial cells are a single cell layer and are important for the enamel formation and mineralization. Wnt/β-catenin signaling has been proven to exert an important role in the mineralization of bone, dentin and cementum. Little was known about the regulatory mechanism of Wnt/β-catenin signaling pathway in ameloblasts during amelogenesis, especially in the mineralization of enamel. To investigate the role of β-catenin in ameloblasts, we established Amelx-Cre; β-catenin^?ex3fl/fl (CA-β-catenin) mice, which could constitutive activate β-catenin in ameloblasts. It showed the delayed mineralization and eventual hypomineralization in the incisor enamel of CA-β-catenin mice. Meanwhile, the amelogenesis-related proteinases Mmp20 and Klk4 were decreased in the incisors of CA-β-catenin mice. These data indicated that β-catenin plays an essential role in differentiation and function of ameloblasts during amelogenesis.     

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.     

Formation of intermediate cementum. II: a scanning electron microscopic study of the epithelial root sheath of Hertwig in monkey

The intermediate cementum is a layer of calcified tissue between the dentin and the cementum at the periphery of dental roots. The mineralization pattern of the intermediate cementum and the innermost layer of aprismatic enamel in the crowns has been shown to be very similar. Since the formation of these tissues is incompletely known and there have been diverging opinions whether they are of epithelial or mesenchymal origin, the present study aimed at investigating the surface morphology of the root sheath with scanning electron microscopy and correlate it to the mineralization of the intermediate cementum. The advancing mineralization front of the intermediate cementum was covered by the root sheath. Numerous microvilli facing the root were found on this part of the root sheath. The corresponding surface facing the dental follicle was covered with bulb-type junctions. Microvilli and bulb-type junctions have also been demonstrated on the surface of the presecretory ameloblast and associated with formation of aprismatic enamel. Thus, the epithelial root sheath seems to actively take part in the formation of intermediate cementum.     

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.     

Immunofluorescent localization of amelogenins in developing bovine teeth.

Antiserum was prepared to the proteins (amelogenins) isolated from fetal bovine enamel matrix. This antiserum was used to localize the amelogenins in the developing bovine molar by immunofluorescent microscopy. Amelogenins could be identified in the preameloblasts before enamel matrix deposition had begun as well as in the secretory ameloblasts. The closely adherent layer of stratum intermedium cells also contained some immunoreactive material, suggesting that they may contribute protein to the enamel matrix. The newly deposited enamel matrix consisted of brightly fluorescent particles. Mature enamel matrix did not contain the immunoreactive protein except in a thin layer along the dentino-enamel junction and adjacent to the ameloblasts. No other portion of the tooth bud or other tissues reacted with the specific antiserum.     

Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization

Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin.     

FAM20C Plays an Essential Role in the Formation of Murine Teeth

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.     

The cadherin-catenin complex is expressed alternately with the adenomatous polyposis coli protein during rat incisor amelogenesis.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, is expressed in highly specific spatiotemporal patterns throughout metazoan development, notably at sites of embryonic induction. E-cadherin also plays a critical role in regulating cell motility/adhesion, cell proliferation, and apoptosis. We have used the continuously erupting rat incisor as a system for examining the expression of E-cadherin and the associated catenins [alpha-, beta-, gamma-catenin (plakoglobin) and p120(ctn)] during amelogenesis. Using immunhistochemical techniques, we observed expression of alpha-catenin and gamma-catenin in ameloblasts throughout amelogenesis. In contrast, expression of E-cadherin, beta-catenin, and p120(ctn) was strong in presecretory, transitional, and reduced stage ameloblasts (Stages I, III, and V) but was dramatically lower in secretory and maturation stage ameloblasts (Stages II and IV). This expression alternates with the expression pattern we previously reported for the adenomatous polyposis coli protein (APC), a tumor suppressor that competes with E-cadherin for binding to beta-catenin. We suggest that alternate expression of APC and the cadherin-catenin complex is critical for the alterations in cell-cell adhesion and other differentiated cellular characteristics, such as cytoskeletal alterations, that are required for the formation of enamel by ameloblasts.     

Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.     

Initial aspects of mineralization at the dentino-enamel junction in embryonic mouse incisor in vivo and in vitro: A tem comparative study

Summary The frontier between the enamel organ and the dental papilla, the future dentino-enamel junction, undergoes coordinated modifications. The mineralization of the extracellular matrix starts within the predentine, which is a prerequisite for the formation of the first enamel crystallites in vivo. We investigated the dentino-enamel junction using the embryonic mouse incisor as a model. Our data showed that the notion of the dentino-enamel junction should not be restricted to the thin interface classically described. A temporo-spatial survey from the epithelio-mesenchymal junction to the dentino-enamel junction delineated a clear sequence of events characterized by the early deposition of electron-dense granules, followed by the appearance of patches of stippled material at the dentino-enamel junction. The first tiny enamel crystallites appeared in the vicinity of this material which presented a well-ordered alignment. The comparison of data obtained in vivo on 17-, 18-, 19-d-old embryonic incisors with those obtained in vitro using 15-d-old embryonic incisors cultured for 7 d emphasizes the relevance of this sequence. Helicoidal growing crystals were observed in cultured tooth germs but never in vivo.