Histological and three dimensional organization of the odontogenic organ in the lower incisor of 100 gram rats.

A three dimensional reconstruction of the epithelial tissue at the apical end of the lower rat incisor was made from serial 1 mum thick cross sections. This tissue formed an elongated structure, called the odontogenic organ, which was composed of a bulbous and a "U"-shaped part. Both parts were joined to one another at the posterior aspect of the apical foramen. The bulbous part of the odontogenic organ was situated at the lingual side of the "U"-shaped part and protruded anteriorly over the pulp. It was formed by cells of the outer dental epithelium and stellate reticulum whose organization suggested that the bulbous part was important in the production of cells for renewal of all the epithelia of the incisor. The "U"-shaped part of the odontogenic organ was apparently derived from the bulbous part and delineated the pulp by forming the lateral, mesial and labial sidewalls around the apical foramen. It was composed of all the epithelial cell types recognizable as precursors to (a) cells of the enamel organ which form the enamel, and (b) Hertwig's epithelial root sheath, a part of the odontogenic organ which induces the formation of dentin on the lingual aspect of the incisor.     

Separation of non-proliferating from proliferating thymic lymphocytes based on light scatter analysis

Summary The migration of the ameloblasts in the continuously erupting incisors of the rat is accompanied by cell loss. Ameloblasts degenerate near the mesial and lateral cemento-enamel junctions in the secretory zone and in the middle two thirds of the region of postsecretory transition, degeneration being most marked where these areas merge. These findings support the hypothesis that the prism decussation in the enamel results from alternating transverse rows of secretory ameloblasts sliding past each other whilst elaborating their rods. The distribution of the degenerating cells suggests, however, that the sliding cell rows are not exactly transverse but arcuate, with the opening facing incisally. The progress of structural alterations of the nuclei in the degenerating ameloblasts appears to follow the pattern earlier described in vinblastine-damaged ameloblasts.     

Regional ultrastructural and cytochemical comparisons of the epithelial-mesenchymal interface during rat incisor development

A major theme in understanding epithelial-mesenchymal interactions during development focuses upon regional mesenchyme specification of epithelial differentiation. One particularly useful epidermal organ system for studying this issue is the rodent continuously growing and erupting incisor tooth organ. One advantage of this particular system resides in the regional features of the rodent incisor tooth organ. Along the labial surface, inner dental epithelial cells differentiate into ameloblasts that produce enamel extracellular matrix, whereas the epithelia along the lingual surface do not become ameloblasts and do not produce enamel matrix. This study has been designed to compare ultrastructural features of labial versus lingual surfaces, with particular emphasis upon mesenchymal cell shape, the orientation of extracellular matrix collagen, the basal lamina, and the distribution of sulfated glycoconjugates. Critical analyses of the data indicated that different microenvironments exist between epithelia and mesenchyme in the labial versus the lingual surfaces of the developing rodent incisor tooth organ.     

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.     

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor

Summary Enzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry.Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts.Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils.Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Summary Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope.Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced K _{1, 2}, x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens.The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Localization of actin during differentiation of the ameloblast, its related epithelial cells and odontoblasts in the rat incisor using NBD-phallacidin

Using NBD-phallacidin, which specifically binds to F-actin, we investigated changes in the localization of actin during the differentiation of ameloblasts, related epithelial cells and odontoblasts in rat incisors. In cryosections treated with NBD-phallacidin, intense fluorescence was observed in undifferentiated epithelial cells in the apical loop and at the proximal extremity of undifferentiated inner enamel epithelial cells. During differentiation, the distal extremity began to exhibit strong fluorescence. In cross-sections of secretory ameloblasts, the fluorescence took the form of polygons of uniform intensity at the proximal end, and of rectangles of non-uniform intensity at the distal end. At the distal end, the fluorescence was more intense at right angles to the long axis of the incisor. At the distal end, this pattern was established just before the appearance of the enamel layer. These patterns were maintained during the secretory stage of ameloblasts. The location, pattern and time of appearance of these sites were identical to those of the terminal webs in ameloblasts. NBD-phallacidin weakly labelled the peripheral cytoplasm of the cell body of ameloblasts, and also labelled Tomes' process. The cells forming the stratum intermedium were mainly labelled at their periphery (i.e. forming larger polygons), while the overlying epithelial cells exhibited labelling throughout their cytoplasm. Except for the terminal webs, the cell bodies of odontoblasts were weakly labelled throughout the period of differentiation. Young odontoblasts secreting predentin were first labelled on the terminal web, with the fluorescence becoming gradually more intense as the thickness of the dentin increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histological and three dimensional organization of the odontogenic organ in the upper incisor of 100 gm rats: comparison with the lower incisor.

The epithelial tissue forming the posterior aspect of the apical foramen in the upper incisor of the rat was reconstructed from 1 mum thick serial cross sections. Like the lower incisor, this portion of the odontogenic organ in the upper incisor was composed of a bulbous and a "U"-shaped part. However, the bulbous part was considerably blunter and the "U"-shaped part much larger in circumference in comparison to the lower incisor. Although no differences were found between the upper and lower incisor regarding the contents and the basic organization of cells within each part of the odontogenic organ, specific differences were found within the bulbous part in the upper incisor. There was a more definitive boundary between the outer dental epithelium and stellate reticulum, a more intimate relationship of cell streams to the stellate reticulum, and a noticeable lack of swirling of cells as part of the streams. These features suggest that the activity inside the bulbous part is less intense in the upper incisor than it is in the lower incisor. In addition, the relationships between the bulbous part, the "U"-shaped part and the root sheath part of the odontogenic organ and the enamel organ were described for the upper incisor.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope. Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced Kalpha1,2 x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens. The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.     

Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.     

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.     

The effect of epidermal growth factor on neonatal incisor differentiation in the mouse

The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine ([3H]TDR) and tritiated proline ([3H]PRO). On days 0 (day of birth), 1, and 2, EGF was administered (3 micrograms/g body wt) sc to neonates. Mice were killed on Days 1, 4, 7, 10, and 13 after birth and were injected with either [3H]TDR or [3H]PRO 1 hr before death. [3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) fibroblasts. [3H]PRO was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [3H]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-microns-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-microns level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 microns, 45 microns,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in vivo 125I-EGF binding was analyzed autoradiographically.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.     

Localization of epidermal growth factor receptors in cells of the enamel organ of the rat incisor.

Epidermal growth factor (EGF) is a peptide shown to effect precocious incisor tooth eruption in rat pups. Binding sites for EGF were visualized in the continuously erupting adult rat incisor by light and electron microscope radioautography after in vivo injection of 125I-EGF. These binding sites represented EGF receptors because of (i) competition between 125I-EGF binding at 2 min after injection and a coinjected excess of unlabeled EGF; (ii) the receptor-mediated endocytosis of 125I-EGF at 15 and 30 min after injection; and (iii) the demonstration of EGF receptor kinase activation in vivo. The stem and the mitotic cells in the epithelial odontogenic organ at the growing end of the tooth develop into two nondividing layers of the enamel organ: (i) ameloblasts which secrete enamel and are subsequently involved in the enamel maturation process, and (ii) papillary layer cells situated between the blood supply and the ameloblasts. Although few EGF receptors were present at the mitotic end, receptor density was highest at the mature end of the enamel organ. High levels of 125I-EGF binding were found on papillary layer cells and ruffle-ended, but not smooth-ended, ameloblasts. This implies a cyclical exteriorization and internalization of receptors during modulations between the two cell types. These data suggest that the EGF receptor mediates a major function of the enamel organ in the formation of enamel.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal''s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.