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The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize., (Copyright 2000 Academic Press.)

The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.

Dendritic cells in the enamel organ of rat incisors were examined with immunocytochemistry using an anti-cystatin C antibody for immature dendritic cells and macrophages, OX6 for MHC Class II, ED1 for macrophages and dendritic cells, and ED2 for macrophages. Single cells positive for anti-cystatin C appeared in the enamel organ in zones at which ameloblasts secrete enamel matrix proteins. They were also present in transition and enamel maturation zones. In addition, ameloblasts, osteocytes, and osteoclasts were labeled by anti-cystatin C. ED1 and ED2 immunocytochemistry revealed that there was no macrophage population in the enamel organ of secretion, transition, or enamel maturation zone. A double labeling study showed that most anti-cystatin C-positive cells in the enamel maturation zone were also positive for OX6, whereas anti-cystatin C-positive and OX6-negative cells were prevalent in the secretion zone. The results suggest that immature dendritic cells penetrate the enamel organ of the secretion zone and begin to mature in the zones of transition and enamel maturation. (J Histochem Cytochem 48:1243-1255, 2000)

Our previous study showed that the migration of terminal Schwann cells occurred in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve (IAN) in the adult animals [Y. Atsumi, K. Matsumoto, M. Sakuda, T. Maeda, K. Kurisu, S. Wakisaka, Altered distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve: An immunohistochemical study on S-100 proteins, Brain Res. 849 (1999) 187-195]. The aim of the present study was to investigate the effects of neonatal transection of the IAN on the regeneration of axon elements and Schwann cells in the periodontal ligament of the rat lower incisor. Following transection of IAN at post-natal day 5 (PN 5d), when the numbers of both axon elements and the terminal Schwann cells were very small, regenerating nerve fibers appeared between post-injured days 7 (PO 7d) and PO 14d, and increased in number thereafter gradually. Although the terminal morphologies of regenerated Ruffini endings became identical to those of the adult animals by PO 54d, the number of regenerated PGP 9.5-IR nerve fibers did not recover the adult levels even by PO 56d. A small number of Schwann cells migrated into the shear zone, the border between the alveolus-related part (ARP) and the tooth-related part (TRP), but did not enter into the TRP. Following transection of the IAN at PN 14d or PN 28d, when clusters of apparent terminal Schwann cells could be recognized, axon regeneration started around PO 5d. Individual axon terminals of the regenerating Ruffini endings ramified and became identical to those of the adult animals around PO 28d, but the number of regenerated Ruffini endings was smaller than that of the adult animals. Similar to the adult animals, the migration of Schwann cells into the shear zone and TRP occurred, and disappeared prior to the completion of the axonal regeneration. The present results indicate that the migration of the Schwann cells into TRP during the regeneration of the periodontal nerve fibers following nerve injury to the IAN depends on the maturation of the terminal Schwann cells of the periodontal Ruffini endings, not on post-operative time.

The internal epithelium of enamel organ and the below enamel surface during growth of the lower incisor, were examinated in ten Wistar rat 12-27 weeks old and weighing between 150/200 gr, by means of immuno histochemical, light and scanning electron microscopy techniques. Our specimens indicate that during the outer enamel secretion the anti-actin positivity goes from distal terminal web to infra nuclear region of cell body. The results of the present study do not support the active movement hypothesis, conversely they support the Warshawsky (1992) hypothesis, i.e. the distal terminal web permits the maintenance and the assembling of ameloblasts during enamel growth. Hence we do agree with Osborn (1970) who reported that, during secretion, ameloblasts move passively in response to secretory forces.

Historically, odontoblasts have been isolated from rat incisor using a surgical curette to separate these cells from the dentin. Isolation of odontoblasts using this approach typically resulted in cells with membrane properties that made the application of patch-clamp electrophysiological techniques prohibitive. The studies here describe a new procedure for isolating mature odontoblasts from adult rat incisor to obtain enriched populations of intact, viable odontoblasts that can be readily studied using patch-clamp methodologies. Identification of isolated cells as odontoblasts was confirmed using in situ mRNA hybridization for expression of dentin sialoprotein, osteocalcin, bone sialoprotein, and type I collagen, and calcium flux was monitored in these cells by means of fura-2 microfluorometry. We suggest that either single odontoblasts or clusters of these cells isolated by this new method would be an ideal preparation for the study of odontoblast properties using electrophysiological techniques, in situ hybridization and/or microfluorometry.

Stromelysin-1 (matrix metalloproteinase-3) or proteoglycanase was visualized by light and electron microscopy immunolabelling in the forming zone of rat incisors. In predentine, labelling was more dense at the transition zone between the inner proximal third and the two outer thirds. Odontoblast processes were also positively stained, mostly in predentine and to a lesser degree in dentine. The dentine-enamel junction was intensely labelled, whereas dentine and forming enamel were only faintly stained. Gold-antibodies complexes were seen inside secretory ameloblasts and odontoblasts in cytosolic locations. The distribution of stromelysin-1 was compared with the distribution of 2-B-6 epitope, an antibody recognizing chondroitin-4-sulphate/dermatan sulphate and which showed a decreasing gradient from the proximal zone to the distal part of predentine. In contrast, both 5-D-4, an anti-keratan sulphate antibody and an anti-lumican antibody displayed a reversed distribution, with an increase seen from the proximal and central thirds to the distal part of predentine. This coordinated distribution suggests that stromelysin-1 may have a functional role, being implicated in predentine in the degradation of chondroitin-4-sulphate/dermatan sulphate-containing proteoglycans, and consequently allowing keratan sulphate proteoglycan concentration to increase near the border where mineralization is initiated.

The continuously growing mouse incisor is an excellent model to analyze the mechanisms for stem cell lineage. We designed an organ culture method for the apical end of the incisor and analyzed the epithelial cell lineage by 5-bromo-2'-deoxyuridine and DiI labeling. Our results indicate that stem cells reside in the cervical loop epithelium consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells, and that they give rise to transit-amplifying progeny differentiating into enamel forming ameloblasts. We identified slowly dividing cells among the Notch1-expressing stellate reticulum cells in specific locations near the basal epithelial cells expressing lunatic fringe, a secretory molecule modulating Notch signaling. It is known from tissue recombination studies that in the mouse incisor the mesenchyme regulates the continuous growth of epithelium. Expression of Fgf-3 and Fgf-10 were restricted to the mesenchyme underlying the basal epithelial cells and the transit-amplifying cells expressing their receptors Fgfr1b and Fgfr2b. When FGF-10 protein was applied with beads on the cultured cervical loop epithelium it stimulated cell proliferation as well as expression of lunatic fringe. We present a model in which FGF signaling from the mesenchyme regulates the Notch pathway in dental epithelial stem cells via stimulation of lunatic fringe expression and, thereby, has a central role in coupling the mitogenesis and fate decision of stem cells.

The X-linked tabby (Ta) syndrome in the mouse is homologous to the hypohidrotic ectodermal dysplasia (HED) in humans. As in humans with HED, Ta mice exhibit hypohidrosis, characteristic defects of hairs and tooth abnormalities. To analyze the effects of Ta mutation on lower incisor development, histology, morphometry and computer-aided 3D reconstructions were combined. We observed that Ta mutation had major consequences for incisor development leading to abnormal tooth size and shape, change in the balance between prospective crown- and root-analog tissues and retarded cytodifferentiations. The decrease in size of Ta incisor was observed at ED13.5 and mainly involved the width of the tooth bud. At ED14.5-15.5, the incisor appeared shorter and narrower in the Ta than in the wild type (WT). Growth alterations affected the diameter to a greater extent than the length of the Ta incisor. From ED14.5, changes in the shape interfered with the medio-lateral asymmetry and alterations in the posterior growth of the cervical loop led to a loss of the labio-lingual asymmetry until ED17.0. Although the enamel organ in Ta incisors was smaller than in the WT, a larger proportion of the dental papilla was covered by preameloblasts-ameloblasts. These changes apparently resulted from reduced development of the lingual part of the enamel organ and might be correlated with a possible heterogeneity in the development of the enamel organ, as demonstrated for upper incisors. Our observations suggest independent development of the labial and lingual parts of the cervical loop. Furthermore, it appeared that the consequences of Ta mutation could not be interpreted only as a delay in tooth development.

First lower E-14 and E-16 mouse molars and E-13 lower incisors were cultured in vitro and either sequentially or continuously labelled with BrdU (5-bromo-2'-deoxyuridine). The behaviour of the non-cycling inner dental epithelial cells emerging from the enamel knot area of the molars was analysed by 3D (three dimensional) reconstructions of serial sections. These cells, as well as slow cycling cells underwent a coordinated temporo-spatial patterning leading to their patchy segregation at the tips of the forming cusps. In incisors (in vitro and in vivo), non-cycling cells were also present in the inner dental epithelium of the enamel knot area. However, these cells were not redistributed during incisor morphogenesis. These non-dividing inner dental epithelium cells of the enamel knot area which are either redistributed or not according to the tooth type specific morphogenesis might represent the organizers of morphogenetic units (OMU), the cusps.

The present study was designed to analyze the structures of dentinal tubules by confocal microscopy. Undecalcified ground sections of human teeth were stained with alizarin red in 0.1% KOH aqueous solution, and examined by confocal microscopy. Alizarin red stained dentinal tubules, interglobular dentine, granular layer of Tomes, and the surface of dentine. Interglobular dentine was seen between the outer and middle layers of coronal dentine. At the outer layer of coronal dentine, the dentinal tubules were thin and showed numerous branches. At the middle layer of coronal dentine, dentinal tubules displayed two types. The type I tubules are the dentinal tubules that do not show any nodular structures and the type II tubules are the dentinal tubules that appear bamboo-like with many nodules. In the cross section through the type II tubules, the nodules appeared as fine circular tubules surrounding the dentinal tubules. The circular tubules of nodules adhered to one side of the dentinal tubules. When the fluorescence images were compared with the images taken by transmission light mode, the fluorescence of dentinal tubules was seen at the inner surface of dentinal tubules, and the fluorescence of nodules was seen at interface between peritubular and intertubular dentine. Most of the dentinal tubules were of the type II tubules in the teeth from older individuals, whereas the type II tubules were scarce in the teeth from younger individuals. At the inner layer of coronal dentine, the dentinal tubules have no nodules and branches were scarce. The dentinal tubules of radicular dentine were different from those of coronal dentine. Most of the dentinal tubules were the type I tubules. Numerous fine branches were seen at the outer and middle layers of radicular dentine. No interglobular dentine was seen in the root except at the cervical part, and the granular layer of Tomes was also positive with alizarin red. At the cervical part of the root, interglobular dentine was present and the dentinal tubules displayed types I and II.

Subcellular localization of S-100 protein, a kind of calcium binding proteins, was examined immunohistochemically in the Ruffini ending, a primary mechanoreceptor, in the periodontal ligament of the rat incisor. The periodontal ligament of the rat incisor was found to contain many S-100beta-immunoreactive (-IR) structures but no S-100alpha-IR elements. The S-100beta-IR structures ramified extensively to form Ruffini endings and were frequently associated with round cells, the terminal Schwann cells, which also showed S-100beta-like immunoreactivity. In many periodontal Ruffini endings, S-100beta-IR products were recognized in the cytoplasm of Schwann cells, but not in the axoplasm. However, some axon terminals which had fewer or shorter axonal fingers, were filled with S-100beta-IR products. The present findings indicated the existence of S-100beta, not S-100alpha, in axon terminals of the periodontal mechanoreceptive endings which were identified as type II Ruffini endings.

Cytochrome oxidase (CO), one of the membrane-bound mitochondrial enzymes involved in oxidative phosphorylation, reflects the functional activity of mitochondria. Mitochondria in the enamel organ show drastic changes in localization during amelogenesis (Smith. INSERM, 1984;125:273-282). In understanding the functional aspects of the enamel organ, it is essential that one knows the exact CO activity in the respective mitochondria. The present study examines the CO activity of mitochondria in the enamel organ of rat incisors throughout the various stages of amelogenesis using light and transmission electron microscopy. CO activity was examined histochemically according to Seligman et al. (J. Cell. Biol., 1968;38:1-14) in decalcified sections of the upper and lower incisors of the rat. In the secretory stage, half of the mitochondria in the ameloblasts accumulated in the infranuclear region were reactive for CO. Both the population and CO activity of the infranuclear mitochondria of ameloblasts decreased significantly in the later stage where the enamel matrix secretion was almost complete. The CO-reactive mitochondria in the cells of the stratum intermedium (SI) gradually increased in number throughout the secretory stage. In the maturation stage, the ameloblasts contained intensively CO-reactive giant mitochondria in the proximal region and regular sized ones in the distal cytoplasm that were mostly devoid of detectable CO reactivity. The proportion of CO-reactive mitochondria in the supranuclear region and the population of mitochondria in the infranuclear regions of the smooth-ended ameloblasts were significantly higher as compared with the respective values in the ruffle-ended ameloblasts. In the late stages of enamel maturation, ameloblasts containing a large number of ferritin-filled pigment vesicles possessed numerous CO-reactive mitochondria between those vesicles in the supranuclear region, implicating an active role of the ameloblasts in iron transfer into the maturing enamel. The papillary layer cells possessed numerous intensively CO-reactive mitochondria throughout the maturation stage. A stage-related variation in the localization of CO-reactive mitochondria in the enamel organ of rat incisors was quantitatively demonstrated. It is conceivable that maturation stage ameloblasts form a functional unit with the papillary layer cells, and operate in energy-requiring events such as active ion transport to, and water and matrix protein removal from the maturating enamel. A sign of such functional integrity among the types of the enamel organ cells (ameloblasts, cells of SI, cells of stellate reticulum, and outer enamel epithelial cells) cannot be seen in the secretory stage. The secretory ameloblasts may function in matrix formation and calcium regulation in a less cooperative manner with the other cells of the enamel organ as compared to the maturation stage ameloblasts.

We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206)., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved)

Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca2+ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca2+ as a second messenger were evident.

The purpose of this study was to use amelogenin as a marker to examine the feasibility of isolating ameloblasts from enamel organ cell populations by fluorescence activated cell sorting. After treating dissected rat enamel organs with proteolytic enzymes to loosen cell attachments and labial connective tissues, dissociated cell suspensions were fixed, then immunostained with rabbit anti-rM179 recombinant amelogenin antibody and FITC-conjugated goat anti-rabbit Ig G antibody. Flow cytometry indicated that about 70% of the total cell sample and virtually all the larger cells therein were amelogenin-positive. Fluorescence activated cell sorting yielded a sample of amelogenin-positive cells at 97% purity. Immunofluorescence microscopy indicated that these isolated amelogenin-positive cells varied widely in size and morphology. This was attributed to loss of intercellular support for ameloblasts once they were dissociated from each other, and to some fragmentation caused when the cells were initially physically removed from the teeth. The results demonstrate that viable ameloblast cell fractions, especially representing cells at the secretory stage, can be purified from enzymic digests of rat enamel organ by sorting on the basis of cell size alone. From these fractions, subpopulations of ameloblasts may be identified when differentiation specific cell surface markers become available.

The cellular distribution of inositol 1,4,5-trisphosphate receptors was examined in rodent maxillary incisor teeth. In situ hybridization studies with a transmembrane probe of type I inositol 1,4,5-trisphosphate receptor indicated that this receptor/channel was highly expressed in odontoblast cells of incisor teeth. In contrast, very low labeling was observed in dental pulp. Northern analysis showed a message size of approximately 9.5 kilobases for this receptor, and demonstrated that type III inositol 1,4,5-trisphosphate receptor was expressed in incisor teeth. Immunocytochemical studies confirmed that types I and III inositol 1,4,5-trisphosphate receptors were both highly expressed in odontoblasts while very low expression was detected in dental pulp. Finally, antibodies that recognized alpha subunits of the Gq class of GTP binding proteins also stained odontoblasts. These results indicate that receptor-mediated regulation of calcium release through inositol 1,4,5-trisphosphate receptors may occur in odontoblasts of rat incisor teeth. These findings also suggest that inositol 1,4,5-trisphosphate receptor/channels regulate calcium flux in odontoblasts during mineralization of dentin, or in growth and differentiation of incisor tissue.

Immunocompetent cells in the enamel organ of rat incisors were examined immunohistochemically using OX6, ED1, and ED2 monoclonal antibodies known to recognize the Class II MHC molecules, a monocyte-macrophage lineage, and residential macrophages, respectively. The OX6 immunopositive cells (MHC cells) were located exclusively in the enamel maturation zone. MHC cells increased in number in the incisal direction and occasionally extended cytoplasmic processes deep into the ameloblast layer. Migration of MHC cells in the ameloblast layer were also encountered. MHC cells lacked phagolysosomes and could be distinguished from typical macrophages. ED2 immunopositive cells were not seen in the enamel organ. ED1 positive cells displayed identical localization to MHC cells except that some appeared in the transitional zone. MHC cells could not be seen in the enamel organ of rat molar tooth germs. Our data confirmed the presence of a large population of "dendritic" immunocompetent cells in the enamel organ of rat incisors and characterized the ultrastructural features of these cells. Biological significance of the immunocompetent cells in the enamel organ during amelogenesis needs to be clarified.

Odontoblasts that we prepared from bovine incisors produced a dentin-specific protein, phosphophoryn, and accumulated it in mineralized nodules. The time course of mineralization was detected by measuring osteocalcin and mineral in the nodules. The sequence of developmental expression of proteins in this mineralizing dentin cell culture is very similar to that in bone cells, suggesting a common mechanism for matrix mineralization in bone and dentin. Casein kinase II, which phosphorylates bone phosphoproteins and dentin phosphorylates bone phosphoproteins and dentin phosphophoryn, also emerges coinciding with the initiation of mineralization. Furthermore, we have detected extracellular phosphorylation by casein kinase II of a dentin protein of M(r) 60,000, which we recovered from the phosphophoryn fraction in CaCl2 precipitate.

The developmental regulation of various receptor forms may be a key-element in the local fine tuning of growth factor effects. The present study focuses on the tissue- and stage-specificity of the alternative splicing of EGF receptor transcripts in the rat incisor. In situ hybridization, as well as light- and electron-microscopic immunolocalization were performed with a set of tools which enable us to discriminate the full-length and secreted truncated forms of EGF receptor. Our data show that, apart from a transient expression in differentiating odontoblasts, EGF receptor expression was predominantly observed in the dental epithelium. In the crown, the expression of the full-length EGF receptor was maximal during preameloblast proliferation and differentiation, decreased in differentiated ameloblasts, and remained low throughout enamel secretion. On the other hand, maturation stage ameloblasts, which regulate the final mineralization of enamel, express high levels of the full-length EGF receptor. In contrast with ameloblasts, epithelial supra-ameloblastic cells, which are not directly involved in the deposition of enamel matrix, showed an alternating predominance of the secreted truncated form during the secretion stage, and the full-length form during the maturation stage. The presence of the secreted truncated EGF receptor form was supported by the electron microscopic detection of extracellular aggregates of immunoreactive EGF receptor. Finally, Northern-blotting of enamel organ samples confirmed the presence of transcripts corresponding to mRNAs of both EGF receptor forms. During root formation, a decreasing gradient of full-length EGF receptor form expression was observed from the apical loop to the disrupting zone in root epithelium. The secreted truncated EGF receptor form was essentially detected in epithelial cells of the disrupting zone of root epithelium. During crown formation, the secreted truncated EGF receptor form, which appears to be synthesized by epithelial supra-ameloblastic cells and secreted toward ameloblasts, may competitively bind EGF receptor ligands and modify activation of the full-length EGF receptor.

Matrix-mediated epithelio-mesenchymal interactions play a crucial role in the control of dental cytodifferentiations. Ultrastructural observation of the epithelio-mesenchymal junction in cultured embryonic mouse molars showed discrete zones with duplicated or multilayered basal laminae. The use of synthetic peptides demonstrated that the process was RGD*-independent, did not involve the YIGSR* sequence present on laminin and could occur spontaneously. Cultured incisors showed a similar but much more dramatic multiplication of the basal laminae. Furthermore, the deposition of multilayered basal laminae was specific for the labial aspect of the tooth and could be detected after 6 h of culture. Despite these alterations, preodontoblasts differentiated and gradients of differentiation were maintained, suggesting that among basement membrane constituents, the basal lamina itself does not play a critical role. More important is the inner dental epithelium which may still control odontoblast differentiation by means of diffusible molecules able to reach surface receptors expressed by preodontoblasts or matrix receptors underlying the basal lamina. Gradients of odontoblast differentiation could result from a progressive acquisition of competence by preodontoblasts.

The relationship between odontoblasts and class II major histocompatibility complex (MHC) antigen-expressing cells in the process of dentinogenesis was studied in rat lower incisors, employing immunohistochemistry using OX6-monoclonal antibody. The dental pulp contained numerous OX6-immunopositive cells that varied in morphology from dendritic to spindle under physiological conditions. Under the electron microscope, these immunopositive cells shared common cytoplasmic features, i.e., multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. At the early stage of dentinogenesis, OX6-immunopositive cells, presumably of the immature type, were located in the subodontoblastic layer. During active dentin formation, the OX6-immunopositive cells increased in number and appeared in the odontoblast layer, associating intimately with fenestrated capillaries situated close to the predentin. These cells showed a dendritic appearance and possessed various sizes of multivesicular bodies and characteristic fine tubulovesicular structures, but never contained typical phagosomes. On the other hand, immunopositive macrophages characterized by typical phagosomes tended to occupy the central portion of the pulp. The results suggest that most, if not all, OX6-immunopositive cells situated deep in the odontoblast layer are dendritic cells playing a role in the defense system of the dental pulp against antigenic molecules arriving from the circulation via the fenestrated capillaries. The increasing number of OX6-immunopositive or immunonegative macrophages appearing near the incisal end of the tooth is thought to be involved in the elimination of degenerated odontoblasts.

The dental papillae isolated from rat incisor were examined morphologically and autoradiographically in order to investigate progressive odontoblast differentiation in vitro. Four to eight days after incubation, the preodontoblasts which were labelled with 3H-thymidine became elongated, polarized, odontoblast-like cells with long cytoplasmic processes. They produced an 3H-proline labelled extracellular matrix on the substrate with which the dental papillae were coated. In addition, the odontoblasts located on the initially formed predentin maintained their ordinary shape as elongated cell bodies with long cytoplasmic processes and polarity fo the organelles during the term of culture. The results indicate that the surface of the agar gel substrate may be a critical requirement for odontoblast differentiation, development, and maturation in the absence of both the basement membrane and the enamel epithelium in the culture system used in this study.

The expression of sulphated glycoprotein-2 (SGP-2) is associated with the onset of cellular atrophy and death in many rodent tissues. This gene has a multifunctional involvement that includes apoptosis, spermatogenesis, promotion of cell-cell interactions, modulation of complement systems and tissue regeneration and remodelling. Using decalcified mandibles, mRNA for SGP-2 in rat incisor tooth ameloblasts was examined by in situ hybridization using 35S riboprobes. The rat incisor is unique in that, at one time, all stages of the complex life cycle of the ameloblasts are represented along the length of the enamel forming aspect of the tooth. The pre-ameloblasts only secrete enamel matrix after mitosis. When the full thickness of the enamel has been formed, a remarkable transition in phenotype takes place in the ameloblast. This transition is accompanied by apoptosis or programmed cell death of approximately 25% of ameloblasts. An additional 25% of ameloblasts undergo apoptosis when maturation of enamel matrix takes place with removal of water and protein from the increasingly mineralized matrix. In the present study, expression of SGP-2 was localized most often in the post-secretory transition and maturation ameloblasts. In contrast, the presecretory and secretory ameloblasts did not demonstrate specific hybridization signals. Consistently, neither the odontoblasts nor the pulp demonstrated hybridization signals. Hence our results support other published results which show that increased expression of SGP-2 is associated with apoptosis. The exact function of the SGP-2 gene and its products is not fully defined.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of IGF-I receptor is reported in the odontogenic epithelium and mesenchyme of the continuously erupting mandibular incisor of the rat by immunohistochemistry using a polyclonal antibody specific to the IGF-I receptor. Odontogenic epithelium is a unique odontogenic sequence in that all stages of the complex life cycle of the ameloblast are represented along the length of the enamel-forming aspect of the tooth. Pre-ameloblasts become post-mitotic before secreting enamel matrix. When the full thickness of the enamel has been formed, a remarkable transition in phenotype takes place in the ameloblast. It changes from a protein secretory cell to one active in maturation of enamel matrix by removal of water and protein from the increasingly mineralized matrix. The distribution and intensity of IGF-I receptor expression varied with the phenotypic stages of the ameloblasts. Diffuse cellular staining for IGF-I receptor was found during the active secretory phase of amelogenesis. However, towards the end of this phase, the staining was confirmed to granular or vesicular structures within the cytoplasm. These granular deposits gradually decreased as the ameloblasts made the transition towards enamel maturation. This transition is accompanied by programmed cell death (apoptosis) of approximately 25% of the ameloblasts and cells in this zone did not stain for IGF-I receptor. With the onset of enamel maturation, diffuse staining of the ameloblast layer was re-established gradually and staining remained evident right up to the reduced enamel epithelium, which joins with the oral epithelium. Strong IGF-I receptor immunoreactivity was observed in the stratum basale and stratum spinosum of the adjacent labial gingival epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemistry and electron microscopic observations on the incisor-tooth organ of transgenic mice expressing the muscle-specific desmin gene under the direction of the vimentin promoter, reveal that the expression of the hybrid transgene occurs both in mesenchymal cells and differentiating odontoblasts. The muscle-specific desmin, as estimated by fluorescence intensity, is more expressed in immature mesenchymal cells than in postmitotic differentiated odontoblasts. The expression of the transgene generates alteration of the odontoblast-intermediate filament network and interferes with the secretory activity of both odontoblasts and ameloblasts. Our results are consistent with the hypothesis that odontoblasts have inductive properties on the differentiation of ameloblasts and that intermediate filaments among other factors play the role of cell and tissue organizer.

Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of non-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75NGFR and p140prototrk) on responsive cells. The continuously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF, proNGF and p75NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75NGFR was correlated with differentiation event only in dental mesenchyme: polarizing odontoblasts expressed p75NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.

Thin cryosections and sections of embedded tissue were prepared from dentine of cryofixed rat incisors. Energy dispersive X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS) have been applied to study the calcium and phosphorus distribution in predentine of these incisors. A small enrichment of calcium and phosphorus was found in the predentine zone near the dentine border. Element distributions were correlated with analyses of the early crystal formation in dentine. These investigations were carried out by parallel applications of electron spectroscopic diffraction (ESD) and electron spectroscopic imaging (ESI) using zero-loss filtering. It was found that the earliest crystal formations already showed the lattice of the hexagonal mineral apatite. They form parallelly arranged chains of dots which coalesce rapidly to form "needle-like" crystallites along the collagen microfibrils.

Transmission electron microscopy of thin sections of the rat incisor pulp revealed that in the middle region of the incisor there were fenestrated capillaries in the "predentinal plexus" and that this region contained the tallest odontoblasts. The odontoblasts gradually became shortened in the incisal part of this region; the fenestrated capillaries in the predentinal plexus changed to continuous type capillaries. Almost all the odontoblasts had degenerated near the incisal end of the tooth. The predentinal plexus disappeared in this region, but the "subodontoblastic capillary plexus" persisted. In a specific region just beneath the worn incisal end, numerous macrophages and polymorphonuclear neutrophils appeared and scavenged the degenerating cells, possibly including the odontoblasts.

Growth factors play an important role in the regulation of cell growth, division and differentiation. In this study the distribution and regulation of insulin-like growth factor-I (IGF-I) in the continuously erupting rat incisor was determined by immunohistochemistry. Results were evaluated both visually and with a computer-based image analysis system. The distribution and intensity of IGF-I immunoreactivity varied with developmental stage of the rat incisor. Strong IGF-I immunoreactivity was observed in differentiating odontoblasts and ameloblasts. The most intense immunoreactivity was observed in secretory ameloblasts, secretory odontoblasts and in maturation ameloblasts. Staining was weak or absent in post-secretory ameloblasts but persisted in post-secretory odontoblasts. Weak to moderate immunoreactivity was also seen in cells of the stratum intermedium and in the reduced enamel epithelium. Surrounding alveolar bone showed strong IGF-I immunoreactivity in osteoblasts and in the stratum basale and stratum spinosum of the adjacent labial gingival epithelium. In order to assess the role of GH in IGF-I expression, GH (65 micrograms/100 g bw) was administered for six days to dwarf GH deficient rats, producing a significant increase in body weight (P < 0.01). Measurements at different stages of odontogenesis showed that the staining intensity of secretory ameloblasts (P < 0.01) and maturation ameloblasts (P < 0.001) was significantly different between untreated and treated animals. These results indicate that IGF-I is present in cell populations of the enamel organ of the rat incisor found previously to exhibit growth hormone receptors, and that expression of IGF is GH dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of macrophages in the lingual periodontal ligament of rat incisors was surveyed by histochemical and immunohistochemical methods. Numerous macrophages showing intense ACPase reactions were located primarily in the shear zone of the periodontal ligament. Immunostaining with an ED1-monoclonal antibody that recognizes various subpopulations of macrophages revealed plentiful positive cells showing flamelike profiles throughout the periodontal ligament, in addition to regular macrophages associated with sinusoidal blood vessels. A similar distribution of flamelike cells expressing Ia antigens was demonstrable with immunostaining using an OX6-monoclonal antibody. A consecutive staining of sections for ACPase histochemistry followed by immunoreactions for Ia antigens revealed the presence of two types of the flamelike cells in the periodontal ligament: one with and the other without distinct ACPase activity, corresponding to the macrophage and the dendritic cell, respectively. Either type of flamelike cells was located in the bone-related and shear zones, whereas only dendritic cells without ACPase activity were restricted to the tooth-related zone. OX6-immunonegative cells showing ACPase reactions were also found in the periphery of the sinusoidal blood vessels. Our data are the first to demonstrate the abundance of macrophages and dendritic cells expressing Ia antigens throughout the lingual periodontal ligament of rat incisors. In addition to regular macrophages, an exclusive localization of macrophages with flamelike extensions has been demonstrated in the bone-related and shear zones of the ligament. The region-specific arrangement of macrophages and dendritic cells with various histochemical and immunological features suggests that the periodontal ligament of rat incisor is a useful model for analyzing the process of differentiation of antigen-presenting cells.

The relationship between odontoblasts and pulp capillaries in the process of dentinogenesis was studied in rat lower incisors, both on the labial and lingual sides, using light and transmission electron microscopy. The odontoblasts showed remarkable differences from the apical to the incisal end. Near the apical end of the tooth, "immature odontoblasts", which were thought to be involved in the formation of the mantle dentin, were arranged in a single layer, and continuous capillaries were located just beneath the odontoblasts. In the middle of the tooth, "mature odontoblasts" with highly developed cell organelles and notable processes formed a pseudostratified layer; fenestrated capillaries were found between these cells close to the predentin. The height of the odontoblast layer and the rate of dentin deposition on the labial (enamel-related) side was significantly greater than that on the lingual (cementum-related) side. Near the incisal end, cementum-related odontoblasts gradually decreased in height and number to become "post-odontoblasts" that produced atubular dentin; continuous capillaries were located subjacent to the post-odontoblasts. On the labial (enamel-related) side, however, odontoblasts retained their pseudostratification; fenestrated capillaries were still observed in the odontoblast layer. No atubular dentin was formed on the labial side.

The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

A nonspecific cholinesterase activity was demonstrated in terminal Schwann cells associated with Ruffini endings in the periodontal ligament of rat incisors at the light and electron microscopic levels. The terminal Schwann cells are ultrastructurally characterized by a well-developed Golgi apparatus and rough endoplasmic reticulum. The cells in this study were positive for nonspecific cholinesterase, whereas ordinary Schwann cells associated with more proximal nerve fibers reacted negatively. The reaction products were densely deposited in the cisternae of the rough endoplasmic reticulum and along the nuclear envelop. A moderately intense labeling was found in the cytoplasmic extensions, in which the reaction products gathered in caveolae and vesicles. These findings indicate that nonspecific cholinesterase is a useful marker to distinguish terminal Schwann cells from ordinary Schwann cells and that the enzyme may be synthesized in the rough endoplasmic reticulum and conveyed toward the axon terminals. Since this enzyme has been known to be shared by the inner bulb of Pacinian corpuscles and the lamellar cells of Meissner's corpuscles, its possible involvement in mechanoreceptive functions in these specialized Schwann cells deserves further investigation.

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.

The enamel organs of rat incisors were separated from the enamel surface and processed for rapid freezing and freeze-substitution. A histochemical stain for calcium (GBHA) of thick Epon sections revealed intense calcium reactions in the secretory ameloblasts, exclusively in the tubulovesicular structures extending throughout their distal cytoplasm. Electron microscopy revealed a thin layer of amorphous material with clusters of electron-dense granules along the distal surface of secretory ameloblasts. In young secretory ameloblasts without typical Tomes' processes, a considerable number of mitochondria were located in the distal cytoplasm and contained numerous electron-dense granules. Similar dense granules as well as fine ribbon-like electron-dense figures, all containing significant amounts of calcium, were observed in some of the tubulovesicular structures at the distal end of these cells. A putative exocytotic figure of such dense granules was also observed. The electron-dense granules were rare in more differentiated ameloblasts with elongated Tomes' processes, which occasionally displayed ribbon-like figures in some of the tubulovesicular structures in the process region. No significant calcium peak was detected in the extracellular amorphous material, secretory granules, or along the lateral plasma membranes. These observations may imply high calcium concentrations in mitochondria and tubulovesicular structures in the distal cytoplasm of secretory ameloblasts relative to that of the cytosol and support the possible contribution of these organelles in secretory ameloblasts to cellular calcium regulation at least in the early stage of amelogenesis.

Localization of junctions between inner enamel-secretory ameloblasts was examined by immunofluorescence microscopy using antibodies against adherens junction proteins, radixin, vinculin, and A-CAM. All antibodies used stained the boundary between the ameloblasts exclusively in the plane where F-actin was abundant. This suggests that the adherens junctions in the ameloblasts are involved in cell-to-cell movement with actin-based microfilament bundles.

The role of glycosaminoglycans and proteoglycans during ontogenesis is not known. The developing tooth offers a potentially important model for studies of structure-function relationships. In this study, we have analysed the temproal and spatial expression of chondroitins of differing sulfation patterns in embryonic molars and incisors. For this purpose, we have used monoclonal antibodies (Mabs) specific for unsulfated, 4-sulfated, and 6-sulfated forms of chondroitin in conjunction with indirect immunofluorescence or immunoperoxidase labeling. Unsulfated chondroitin was not detected in embryonic teeth. Chondroitin 4- and chondroitin 6-sulfates were present in the stellate reticulum but otherwise they were confined to the dental mesenchyme. The 3B3 and MC21C-epitope, which are markers of 6-sulfated chondroitin, were uniformly distributed in the dental mesenchyme during the bud stage; they disappeared from the dental papilla of the cusps and of the anterior region of the incisor as development proceeded. These epitopes were absent from the basement membrane and from the predentin. In the odontoblastic cell lineage, the 3B3 and MC21C-epitopes were detected only between preodontoblasts at an early stage of differentiation. The monoclonal antibody 2B6 served as a probe to localize chondroitin 4-sulfate. This glycosaminoglycan was detected as early as the dental lamina stage but its expression was restricted to the basement membrane of the teeth until the late bell stage. After the onset of cusp formation, strong staining was also observed over the occlusal region of the dental papilla while the cervical region of the dental papilla remained 2B6-negative. Incisors at the bell stage exhibited a decreasing gradient of immunostaining by 2B6 from their anterior region to their posterior end. The extracellular matrix surrounding preodontoblasts reacted with 2B6 and the predentin, produced by the odontoblasts, was also intensely labeled with this antibody. Comparison between immunostaining with 3B3 and 2B6, on consecutive sections revealed a mutually exclusive pattern of distribution of the corresponding epitopes during odontogenesis. Furthermore, in the continuously growing incisor, a striking positive correlation was found between the immunostaining patterns produced by 3B3 and MC21C and the mitotic indices along the anterior-posterior axis of the tooth. Hence, sulfation of chondroitin seems developmentally regulated. We postulate that changes in the sulfation pattern of chondroitin might play a role in ontogenesis by locally altering the functional properties of the extracellular matrix.

Conditions of formation of induced osteo-hemopoietic foci in mice, in response to ectopic implantation to them of homologous demineralized tooth matrix, have been studied. It is shown that the hemopoietic tissue of the induced foci is similar to the bone marrow of the skeletal bones by the morphological parameters and cellular composition; differentiation of precursor cells takes place in all three directions of myelopoiesis. Induced foci formation under the capsule of the kidney occurs much later (after 5-8 months) than under the skin of muscular fascia (1-1.5 months). The induced hemopoietic foci functioning continues at least for one year.

Sixty female Wistar rats were injected with tritiated thymidine and killed at intervals between 1 hr and 72 hr after injection. Autoradiographs of paraffin and Vestopal W embedded sagittal sections of the maxillary incisors were prepared. The distribution of labeled cells after 1 hr revealed the basal pulp cells and the preodontoblasts as proliferative cells. The growth rate of the incisor and the migration of the basal pulp cells were established from the position of labeled cells at 72 hr observation time. The number and localization of labeled and unlabeled mitoses were registered in order to determine the duration of the generation cycle of the proliferative cells. The identical cell generation time of preodontoblasts and basal pulp cells and the migration pattern of the latter indicate that the preodontoblasts are renewed by preodontoblast multiplication and not by migration of basal pulp cells.

The effect of adriamycin on rat incisor was investigated 1 day after administration. Rats were injected intravenously with adriamycin at a dose of 5 mg/kg body weight and sacrificed by perfusion with a 2.5% phosphate buffered glutaraldehyde solution. The principal effect of the drug on the incisor was the production of cell degeneration. This was extensive in the apical region, being present along the entire periphery of the dental papilla. In sections examined more incisally, cell degeneration gradually disappeared from the labial portion of the incisor but was present in the lingual portion. This degeneration of cells was not present at the site where mature odontoblasts had differentiated on the lingual surface of the pulp chamber. It appears that the affected cells were early preodontoblasts and the precursors of preodontoblasts. However, as these cells became more differentiated they apparently became more resistant to the drug's effect.

Patterns characteristic of enamel maturation can be visualized at the surface of the rat incisor by staining with glyoxal bis(2-hydroxyanil) (GBHA) and radioautography following 45calcium injection. In this study, the effects of vinblastine on enamel maturation were monitored by these two methods. At 4 h after injection of vinblastine, the darkly-stained GBHA bands had widened incisally into the interband regions when compared to normal, control teeth. Radioautography at 5 min after calcium injection in vinblastine-treated animals (4 h) showed a modified maturation pattern of weaker labeling and less distinct banding. At 8 h after vinblastine injection, most of the enamel stained uniformly with GBHA, and bands and interband regions could not be resolved. Radioautography at 5 min after calcium injection showed that the 8 h vinblastine treatment removed the banding pattern, leaving only a weakly-labeled area. Vinblastine is known to destroy and prevent the formation and turnover of microtubules, and hence the formation of ruffled borders of ruffle-ended ameloblasts (Akita et al. 1983). The concomitant decrease in calcium incorporation implies that events taking place in relation to the ruffled border may affect calcium exchange or accretion within the enamel.

Rats were injected with Calcein and killed 1 h, and 1, 2 and 3 days after injection. Incisors were stained with glyoxal-bis-(2-hydroxyanil) (GBHA) and the enamel surface examined in a fluorescence microscope with incident-light excitation. In maturing enamel Calcein produced five fluorescent double bands across the long axis of the tooth. GBHA produced a series of single red stripes, each of which was imposed on a double Calcein band 1 h after injection. At longer intervals after injection GBHA stripes were seen to be gradually displaced in the apical direction from the Calcein double bands. Since GBHA is known to stain enamel adjacent to smooth-ended ameloblasts this supports the view of a cyclic modulation of ameloblast morphology and function.

Turnover of cells within the odontogenic organ was studied in three dimensions by preparing serial sections of incisors from young male rats killed at various times following a single intraperitoneal injection of 1 muCi/g body weight of 3H-thymidine. Radioautographs showed that at 1 hour after injection labeled cells were present in all cell layers throughout the entire depth of the odontogenic organ. They were encountered frequently within the inner dental epithelium and stratum intermedium but appeared less abundant within the stellate reticulum and outer dental epithelium. With time, the frequency of labeled cells in each layer declined progressively, and more rapidly at the anterior and labial side of the odontogenic organ than toward its posterior and lingual side. Hence labeled cells were observed over the longest time interval in regions where cell layers were in closest proximity to the opening of the apical foramen, that is, near the apical and cervical loops. By 32 days after injection, numerous labeled cells could still be identified within the outer dental epithelium and stellate reticulum near the apical loop (bulbous part of the odontogenic organ) and the outer dental epithelium near the cervical loops ("U"-shaped part of the odontogenic organ). These findings support the hypothesis that cells originate within the bulbous part of the odontogenic organ and migrate anteriorly through the "U"-shaped and root sheath parts of the odontogenic organ during renewal of the incisor. It appears that individual stem cell compartments may be maintained for surface (outer/inner dental epithelium) and intermediate layers (stellate reticulum/stratum intermedium) in the odontogenic epithelium.

Rat incisors cleaned of overlying enamel organ cells stain unevenly with several common histological stains producing two kinds of banding pattern. In one a pattern of widely spaced stained bands run in an oblique to transverse direction from a more apical level on the medial side to a more incisal level on the lateral side of each tooth. The pattern correlated directly with that produced by both in vivo and in vitro short-term tetracycline labelling in the same teeth: it also resembled the patterns previously demonstrated in a) horseradish peroxidase penetration into lateral intercellular spaces between maturation ameloblasts, b) the distribution of enamel labelling in vivo with 45Ca, c) of etching of enamel surfaces demineralized with EDTA with the glutaraldehyde fixed enamel organ in situ and d) in the distribution of smooth-ended and ruffled border types of cell specializations. We conclude that these bands demonstrate further and previously unrecognised aspects of the cyclical phenomena in enamel maturation-mineralization and show cyclical differences in the physico-chemical status of the organic matrix in the maturation process. A second, incremental growth type of banding pattern occurred at 200-225 micron intervals in the lower incisors and had the same distribution at the beginning and end of the maturation zone.